Compositions and methods of chimeric autoantibody receptor T cells

ABSTRACT

The invention includes compositions comprising at least one chimeric autoantibody receptor (CAAR) specific for an autoantibody, vectors comprising the same, compositions comprising CAAR vectors packaged in viral particles, and recombinant T cells comprising the CAAR. The invention also includes methods of making a genetically modified T cell expressing a CAAR (CAART) wherein the expressed CAAR comprises a desmoglein extracellular domain.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of, and claims priority to, U.S. patentapplication Ser. No. 15/307,644, filed Oct. 28, 2016, now allowed, whichis a 35 U.S.C. § 371 national phase application from, and claimspriority to, International Application No. PCT/US2015/028872, filed May1, 2015, and published under PCT Article 21(2) in English, which claimspriority to and the benefit of U.S. Provisional Application No.61/987,989, filed May 2, 2014, all of which applications areincorporated herein by reference in their entireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under AR057001, awardedby the National Institutes of Health. The government has certain rightsin the invention.

BACKGROUND OF THE INVENTION

Autoimmunity is the third most common category of disease in the UnitedStates, affecting 8% of the population. There are two basic categoriesof autoimmune diseases: those predominantly caused by T cells, and thosepredominantly caused by B cells and the autoantibodies they produce.Pemphigus vulgaris (PV) is a model autoantibody-mediated disease, inwhich autoantibodies against the skin cell adhesion protein desmoglein 3(Dsg3) cause potentially fatal blistering of the skin and mucousmembranes.

Current therapies focus on general immune suppression to reduce allantibodies, but these strategies also target good antibodies thatprotect us from infection. Because pemphigus is a chronicremitting-relapsing disease, such treatments are associated withmultiple side effects, including risk of fatal infection and secondarycancers. As an example, rituximab, an anti-CD20 monoclonal antibodyreagent, has been reported to have excellent efficacy in the treatmentof pemphigus vulgaris, with with 95% of patients achieving completehealing of blisters within 3 months, and 35% of patients achievingcomplete remission off all systemic therapies during long term followup. However, greater than 80% of patients will relapse (presumably sincethe efficacy of CD20+ B cell depletion by rituximab is usuallyincomplete), and serious infections are not uncommon, reported to occurin 7% of autoimmune disease patients treated with rituximab, with fatalinfection in 1-2%. Therefore, patients with severe autoimmune diseases,such as pemphigus vulgaris, paraneoplastic pemphigus or pemphigusfoliaceus, are no longer dying from their disease, but instead aresuffering from complications of treatment.

However, therapeutic strategies for the treatment of PV to target onlythe autoreactive B cells do not currently exist. Systemiccorticosteroids, azathioprine, mycophenolate mofetil andcyclophosphamide are effective in the treatment of PV, butnon-specifically inhibit lymphocyte proliferation. Rituximab targetsCD20 expressed on most B cells, but lacks specificity to only theautoreactive B cells.

As a result, therapeutic strategies can pose serious side effectsrelated to general immune suppression, including fatal infection andsecondary cancers. Therefore, a need exists for a therapy that targetsonly the autoreactive B cells.

SUMMARY OF THE INVENTION

As described below, the present invention includes compositions of andmethods for their use, of a chimeric autoantibody receptor (CAAR)specific for an autoantibody.

One aspect of the invention includes an isolated nucleic acid sequenceencoding a chimeric autoantibody receptor (CAAR), wherein the isolatednucleic acid sequence comprises a nucleic acid sequence of anautoantigen or fragment thereof, a nucleic acid sequence of atransmembrane domain, a nucleic acid sequence of an intracellular domainof a costimulatory molecule, and a nucleic acid sequence of a signalingdomain.

In another aspect, the invention includes a vector comprising theisolated nucleic acid sequence described herein.

In still another aspect, the invention includes an isolated chimericautoantibody receptor (CAAR) comprising an extracellular domaincomprising an autoantigen or fragment thereof, a transmembrane domain,and an intracellular signaling domain.

In yet another aspect, the invention includes a genetically modifiedcell comprising the CAAR described herein.

In various embodiments of the above aspects or any other aspect of theinvention delineated herein, the autoantigen is selected from the groupconsisting of Dsg1, Dsg3, and a fragment thereof. In one embodiment, theautoantigen comprises Dsg3 comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ IDNO:11, SEQ ID NO:12, and SEQ ID NO:36.

In another embodiment, the autoantigen comprises Dsg3 and the isolatednucleic acid sequence further comprises a nucleic acid sequence encodinga propeptide of Dsg3. In some embodiments that include the Dsg3propeptide, it comprises an amino acid sequence of SEQ ID NO:2.

In another embodiment, the isolated nucleic acid sequence furthercomprises a nucleic acid sequence of a CD8 alpha chain signal peptide.In some embodiments that include the CD8 alpha chain signal peptide, itcomprises an amino acid sequence of SEQ ID NO:1.

In yet another embodiment, the nucleic acid sequence of thetransmembrane domain encodes a CD8 alpha chain hinge and transmembranedomain. In some embodiments that include the CD8 alpha chain hinge andtransmembrane domain, the transmembrane domain comprises an amino acidsequence of SEQ ID NO:13.

In still another embodiment, the isolated nucleic acid sequence furthercomprises a nucleic acid sequence of a peptide linker. In someembodiments that include the peptide linker, it comprises an amino acidsequence of SEQ ID NO:14.

In another embodiment, the nucleic acid sequence of the intracellularsignaling domain comprises a nucleic acid sequence encoding a CD137intracellular domain. In some embodiments that include the CD137intracellular domain, the intracellular signaling domain comprises anamino acid sequence of SEQ ID NO:15.

In yet another embodiment, the nucleic acid sequence of theintracellular signaling domain comprises a nucleic acid sequenceencoding a CD3 zeta signaling domain. In some embodiments that includethe CD3 zeta signaling domain, the intracellular signaling domaincomprises an amino acid sequence of SEQ ID NO:16.

In one embodiment, the cell comprising the CAAR, expresses it and hashigh affinity to autoantibodies expressed on B cells. In anotherembodiment, the cell expresses the CAAR and induces killing of B cellsexpressing autoantibodies. In still another embodiment, the cellexpresses the CAAR and has low affinity to antibodies bound to a Fcreceptor. In yet another embodiment, the cell expresses the CAAR and haslimited toxicity toward healthy cells. In another embodiment, the cellis selected from the group consisting of a helper T cell, a cytotoxic Tcell, a memory T cell, regulatory T cell, gamma delta T cell, naturalkiller (NK) cell, cytokine induced killer cell, a cell line thereof, andother effector cell.

In another aspect, the invention includes a method for treating anautoimmune disease in a subject, the method comprising: administering tothe subject an effective amount of a genetically modified T cellcomprising an isolated nucleic acid sequence encoding a chimericautoantibody receptor (CAAR), wherein the isolated nucleic acid sequencecomprises an extracellular domain comprising an autoantigen or fragmentthereof, a nucleic acid sequence of a transmembrane domain, and anucleic acid sequence of an intracellular signaling domain, therebytreating the autoimmune disease in the subject. The autoimmune diseaseincludes pemphigus vulgaris, paraneoplastic pemphigus, and pemphigusfoliaceus.

In various embodiments of the above aspects or any other aspect of theinvention delineated herein, the subject is a human. In anotherembodiment, the modified T cell targets a B cell.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of preferred embodiments of theinvention will be better understood when read in conjunction with theappended drawings. For the purpose of illustrating the invention, thereare shown in the drawings embodiments which are presently preferred. Itshould be understood, however, that the invention is not limited to theprecise arrangements and instrumentalities of the embodiments shown inthe drawings.

FIG. 1 is a schematic drawing that depicts how the proposed chimericautoantibody receptor (CAAR) is distinct from all previously developedtechnologies.

FIG. 2 is an illustration showing that engineered chimeric T cellreceptors target Dsg3 specific B cells.

FIG. 3 is an illustration showing targeting of Dsg specific memory Bcells and removing short-lived antibody-secreting B cells.

FIG. 4 is a schematic drawing of the protein domains comprising adesmoglein 3 (Dsg3) chimeric autoantibody receptor (CAAR).

FIG. 5 is an image showing the amplification of the individual domainsused in the Dsg3 CAAR from cDNA of peripheral blood mononuclear cells.

FIG. 6 is an image showing the amplification of CD137 used in the Dsg3CAAR from cDNA of peripheral blood mononuclear cells.

FIG. 7 is a set of images showing the amplification of Dsg3 used in theDsg3 CAAR from plasmid DN653.

FIG. 8 is an image showing a western blot of Dsg3 CAAR to determineprotein expression 48 hours after transformation and cell lysis of 293Tcells under reducing conditions. Dsg3 E-His baculovirus supernatant is apositive control, untransfected HEK293T cells are a negative control.The expected size is 96 kDa for the unglycosylated protein, whichtypically migrates at ˜112 kD with glycosylation.

FIG. 9 is a schematic drawing describing the experiments to testspecificity of the Dsg3 CAAR toward intended and unintended targets.

FIG. 10 is a panel of flow cytometry plots showing the Dsg3 CAAR signalsafter exposure to the intended target. NFAT-GFP Jurkats cells expressingDsg3-CAAR were stimulated with antibody coated beads at a ratio of 3:1(beads:cells). AK23, PV4B3, and PV2B7 are Dsg3-specific mAbs, which ifbound to the CAAR, should trigger signaling resulting in GFP expression.EF1a promoter functioned better than the PGK promoter and resulted inspecific signaling. SS1=anti-mesothelin CAR, positive control, hadbaseline positive activity. Non-transduced, negative-control, no GFPsignal detected.

FIG. 11 is a panel of flow cytometry plots showing the Dsg3 CAAR induceslow level but specific signaling after exposure to polyclonal pemphigusvulgaris (PV) patient serum IgG (reflecting the low overall percentageof total IgG that is Dsg3-specific).

FIG. 12 is a panel of flow cytometry plots showing the Dsg3 CAARresponds to low numbers of surface IgG+ cells (AK23 hybridoma) in adose-dependent manner.

FIG. 13 is a panel of flow cytometry plots showing the safety of Dsg3CAARs by no induction of signaling when exposed to Dsg3 expressingkeratinocytes, indicating that interactions of Dsg3 with desmosomalcadherins on keratinocytes should not result in skin or mucous membranetoxicity.

FIG. 14 is a schematic diagram showing the different scenarios fortesting cytotoxicity toward target cells that express anti-Dsg3 surfaceautoantibodies and off-target cells that express surface Fc receptorsthat could bind serum PV autoantibodies resulting in unintendedredirected lysis.

FIG. 15 is a panel of graphs showing Dsg3 CAAR does not demonstrateredirected lysis against a K562 cell line that expresses surface Fcreceptors that are pre-loaded with PV anti-Dsg3 mAb (PV2B7).

FIG. 16A is an image of an electrophoretic gel showing amplification ofthe different Dsg3 extracellular domains, EC2-3, EC1-2, EC1-3, EC1-4 andEC1-5, which are constructed to optimize Dsg3 CAAR cytotoxicity, sincethe efficacy of CAAR-mediated cytotoxicity is dependent on the distancebetween effector and target cell.

FIG. 16B is a schematic drawing of the Dsg3 extracellular domainsamplified in FIG. 16A.

FIG. 17 is a panel of flow cytometry plots showing the Dsg EC1-3, EC1-4,EC1-5 CAARs can be expressed in primary human T cells and are recognizedby 3 different PV anti-Dsg3 mAbs, AK23, Px44, and F779. EC1-2 did noteffectively express. 21D4=neg control CAR.

FIG. 18 is a panel of graphs showing the efficacy of the Dsg3 CAARagainst an anti-Dsg3 IgG mouse hybridoma (meant to model a PV-specifichuman memory B cell or plasmablast that displays anti-Dsg3 IgG on thecell surface). The Dsg3 CAAR, expressed on the surface of primary humanT cells, shows specific in vitro killing of AK23 (an anti-Dsg3hybridoma) in a chromium release assay after 4 hours.

FIG. 19 is a panel of graphs showing Dsg3 CAAR killing of AK23 hybridomaincreases over time in a chromium release assay after 24 hours. Thekilling efficacy of the CAARs correlates with CAAR size (shorter isbetter so EC1-3>EC1-4>EC1-5). Mock=anti-human CD19 CAR; human CD19 isnot expressed on the target hybridoma cell. Control hybridoma (BK2)which does not express a Dsg3-autoantibody, shows some killing by Dsg3CAARs over 24 hours perhaps due to human-mouse alloreactivity.

FIG. 20 is a graph showing killing of anti-Dsg3 cells targetingdifferent Dsg3 epitopes by Dsg3 CAART cells in a 4 hour chromium releaseassay. Values are representative of at least 4 experiments with T cellsfrom different normal donors (ND).

FIG. 21 is a graph showing killing of anti-Dsg3 cells targetingadditional different human Dsg3 epitopes by Dsg3 CAART cells in a 4 hourchromium release assay.

FIG. 22 is a panel of images showing that surface IgG density ofanti-Dsg hybridomas is comparable to human memory B cells. Differentcell types are taken into account to calculate the relative targetdensity.

FIG. 23 is a graph showing relative affinity ELISA for different targetantibodies of the Dsg3 CAAR, indicating that Dsg3 CAART cells killtarget cells within a range of antibody affinities.

FIG. 24 is a panel of graphs showing that Dsg3 CAART cells are killedeven in the presence of soluble blocking anti-Dsg3 antibody. Effector totarget (E:T) ratio for all conditions: 30:1. ⁵¹Cr, 8 hours.

FIG. 25 is a panel of graphs showing that Dsg3 CAART cells do not killFc receptor expressing cells that may bind soluble anti-Dsg3 antibodiesin PV patient serum by reversed antibody-dependent cellular toxicity(rADCC) in vitro.

FIG. 26 is a graph showing that Dsg3 CAART cells do not kill primarykeratinocytes.

FIG. 27 is a graph showing that Dsg3 CAAR T cells effectively controlbioluminescent IgG secreting anti-Dsg3 hybridoma cells in vivo.

FIG. 28 is a panel of graphs showing by flow cytometric analysis that inthe 4 mice from FIG. 27 that escape Dsg3 CAART treatment, bioluminescent“escape” hybridomas do not express surface IgG, explaining why they areno longer targeted by Dsg3 CAART cells. The numbers on the 3 panels onthe right indicate individual mice. 9406 and 9407 show GFP+ cells thatare surface IgG negative, indicating that these cells were not targetedby the Dsg3 CAAR T cells.

FIG. 29 is a graph showing the presence and engraftment of Dsg3 CAAR Tcells transplanted into mice.

FIG. 30A is a graph showing presence of Dsg3 CAAR T cells in blood.

FIG. 30B is a graph showing presence of Dsg3 CAAR T cells in bonemarrow.

FIG. 30C is a graph showing presence of Dsg3 CAAR T cells in spleen.

FIG. 31 is a graph showing that Dsg CAAR T cells did not cause rADCCagainst FcgammaR-expressing neutrophils and monocytes in vivo. Referencevalues are indicated by horizontal lines. NSG mice do not have B, Tlymphocytes/NK cells.

FIG. 32A is a graph showing bioluminescence of AK23 tumor burden in Dsg3EC1-3 CAART injected mice.

FIG. 32B is a graph showing survival of control and Dsg3 EC1-3 CAARTinjected AK23 tumor bearing mice.

FIG. 33 is a panel of graphs showing that Dsg3 EC1-4 CAAR T cellsexhibit efficacy against a broad range of targets in vivo(bioluminescence imaging days 0-3 after injection).

FIG. 34 is a panel of graphs showing Dsg3 EC1-4 CAAR T cells exhibitefficacy against a broad range of targets in vivo (bioluminescenceimaging days 7-13 after injection).

FIG. 35 is a panel of graphs showing Dsg3 EC1-4 CAAR T cells exhibitefficacy against a broad range of targets in vivo, based on survivalcurves with total flux of 10E8 defined as death.

FIG. 36 is a schematic drawing of the protein domains comprising adesmoglein 1 (Dsg1) chimeric autoantibody receptor (CAAR).

FIG. 37A is a panel of graphs showing killing of two different anti-Dsg1cells by Dsg1 CAART cells by targeting both EC1 and EC2 domains.

FIG. 37B is a panel of graphs showing that Dsg1 CAART cells do notnon-specifically kill wild type K562 cells or K562 cells expressinganti-Dsg3 antibody PVB28.

FIG. 38 is a schematic drawing of the KIR domains in a desmoglein 1 or 3chimeric autoantibody receptor (CAAR).

FIG. 39A is a graph showing killing of anti-Dsg3 (PVB28 anti-EC2) cellsby Dsg3EC1-3 and Dsg3 EC1-4 KIR-CAART cells in a 16 hour chromiumrelease assay.

FIG. 39B is a graph showing that no killing of wild type K562 cells bythe Dsg3 KIR CAARs occurs.

DETAILED DESCRIPTION

The invention includes compositions comprising at least one chimericautoantibody receptor (CAAR) specific for an autoantibody, vectorscomprising the same, compositions comprising CAAR vectors packaged inviral particles, and recombinant T cells comprising the CAAR. Theinvention also includes methods of making a genetically modified T cellexpressing a CAAR (CAART) wherein the expressed CAAR comprises adesmoglein extracellular domain.

The present invention also relates generally to the use of T cellsengineered to express a Chimeric AutoAntibody Receptor (CAAR) to treatan autoimmune disease associated with expression of self-antigens. Inone embodiment, the T cells expressing the CAAR of the inventionspecifically bind to and kill desmoglein 1 or 3 autoantibody expressingcells, but not normal antibody expressing cells.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice of and/or for the testing of the present invention, thepreferred materials and methods are described herein. In describing andclaiming the present invention, the following terminology will be usedaccording to how it is defined, where a definition is provided.

It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

“About” as used herein when referring to a measurable value such as anamount, a temporal duration, and the like, is meant to encompassvariations of ±20% or ±10%, in some instances ±5%, in some instances±1%, and in some instance ±0.1% from the specified value, as suchvariations are appropriate to perform the disclosed methods.

The term “antibody,” as used herein, refers to an immunoglobulinmolecule binds with an antigen. Antibodies can be intact immunoglobulinsderived from natural sources or from recombinant sources and can beimmunoreactive portions of intact immunoglobulins. Antibodies aretypically tetramers of immunoglobulin molecules. The antibody in thepresent invention may exist in a variety of forms where the antibody isexpressed as part of a contiguous polypeptide chain including, forexample, a single domain antibody fragment (sdAb), a single chainantibody (scFv) and a humanized antibody (Harlow et al., 1999, In: UsingAntibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, ColdSpring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci.USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).

The term “high affinity” as used herein refers to high specificity inbinding or interacting or attraction of one molecule to a targetmolecule.

The term “antigen” or “Ag” as used herein is defined as a molecule thatprovokes an immune response. This immune response may involve eitherantibody production, or the activation of specificimmunologically-competent cells, or both. The skilled artisan willunderstand that any macromolecule, including virtually all proteins orpeptides, can serve as an antigen. Furthermore, antigens can be derivedfrom recombinant or genomic DNA. A skilled artisan will understand thatany DNA, which comprises a nucleotide sequences or a partial nucleotidesequence encoding a protein that elicits an immune response thereforeencodes an “antigen” as that term is used herein. Furthermore, oneskilled in the art will understand that an antigen need not be encodedsolely by a full length nucleotide sequence of a gene. It is readilyapparent that the present invention includes, but is not limited to, theuse of partial nucleotide sequences of more than one gene and that thesenucleotide sequences are arranged in various combinations to encodepolypeptides that elicit the desired immune response. Moreover, askilled artisan will understand that an antigen need not be encoded by a“gene” at all. It is readily apparent that an antigen can be generatedsynthesized or can be derived from a biological sample. Such abiological sample can include, but is not limited to a tissue sample, atumor sample, a cell or a biological fluid.

By “autoantigen” is meant an endogenous antigen that stimulatesproduction of an autoimmune response, such as production ofautoantibodies. Autoantigen also includes a self-antigen or antigen froma normal tissue that is the target of a cell-mediated or anantibody-mediated immune response that may result in the development ofan autoimmune disease. Examples of autoantigens include, but are notlimited to, desmoglein 1, desmoglein 3, and fragments thereof.

The term “limited toxicity” as used herein, refers to the peptides,polynucleotides, cells and/or antibodies of the invention manifesting alack of substantially negative biological effects, anti-tumor effects,or substantially negative physiological symptoms toward a healthy cell,non-tumor cell, non-diseased cell, non-target cell or population of suchcells either in vitro or in vivo.

“Autoantibody” refers to an antibody that is produced by a B cellspecific for an autoantigen.

The term “autoimmune disease” as used herein is defined as a disorder orcondition that results from an antibody mediated autoimmune responseagainst autoantigens. An autoimmune disease results in the production ofautoantibodies that are inappropriately produced and/or excessivelyproduced to a self-antigen or autoantigen.

As used herein, the term “autologous” is meant to refer to any materialderived from the same individual to which it is later to bere-introduced into the individual.

“Allogeneic” refers to a graft derived from a different animal of thesame species.

“Xenogeneic” refers to a graft derived from an animal of a differentspecies.

“Chimeric autoantibody receptor” or “CAAR” refers to an engineeredreceptor that is expressed on a T cell or any other effector cell typecapable of cell-mediated cytotoxicity. The CAAR includes an antigen orfragment thereof that is specific for a pathogenic autoantibody. TheCAAR also includes a transmembrane domain, an intracellular domain and asignaling domain.

As used herein, the term “conservative sequence modifications” isintended to refer to amino acid modifications that do not significantlyaffect or alter the binding characteristics of the antibody containingthe amino acid sequence. Such conservative modifications include aminoacid substitutions, additions and deletions. Modifications can beintroduced into an antibody of the invention by standard techniquesknown in the art, such as site-directed mutagenesis and PCR-mediatedmutagenesis. Conservative amino acid substitutions are ones in which theamino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine), beta-branchedside chains (e.g., threonine, valine, isoleucine) and aromatic sidechains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, forexample, one or more amino acid residues within the extracellularregions of the CAAR of the invention can be replaced with other aminoacid residues having a similar side chain or charge and the altered CAARcan be tested for the ability to bind autoantibodies using thefunctional assays described herein.

“Co-stimulatory ligand,” as the term is used herein, includes a moleculeon an antigen presenting cell (e.g., an aAPC, dendritic cell, B cell,and the like) that specifically binds a cognate co-stimulatory moleculeon a T cell, thereby providing a signal which, in addition to theprimary signal provided by, for instance, binding of a TCR/CD3 complexwith an MHC molecule loaded with peptide, mediates a T cell response,including, but not limited to, proliferation, activation,differentiation, and the like.

A “co-stimulatory molecule” refers to the cognate binding partner on a Tcell that specifically binds with a co-stimulatory ligand, therebymediating a co-stimulatory response by the T cell, such as, but notlimited to, proliferation. Co-stimulatory molecules include, but are notlimited to an MHC class I molecule, BTLA and a Toll ligand receptor.

“Desmoglein 1” or “Dsg1” refers to a calcium binding transmembraneglycoprotein that is a component of desmosomes found in cell-celljunctions between epithelial cells. An exemplary Dsg1 sequence includeshuman Dsg1 found at GenBank Accession No. NM_001942 and NP_001932, or afragment thereof, and the mouse Dsg1 sequence found at NM_010079 orNP_034209, or a fragment thereof.

“Desmoglein 3” or “Dsg3” refers to a calcium binding transmembraneglycoprotein that is a component of desmosomes found in cell-celljunctions between epithelial cells. An exemplary Dsg3 sequence includeshuman Dsg3 found at GenBank Accession No. NM_001944 and NP_001935(P32926), or a fragment thereof, and the mouse Dsg3 sequence found atNM_030596 or NP_085099, or a fragment thereof.

“Encoding” refers to the inherent property of specific sequences ofnucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, toserve as templates for synthesis of other polymers and macromolecules inbiological processes having either a defined sequence of nucleotides(i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and thebiological properties resulting therefrom. Thus, a gene encodes aprotein if transcription and translation of mRNA corresponding to thatgene produces the protein in a cell or other biological system. Both thecoding strand, the nucleotide sequence of which is identical to the mRNAsequence and is usually provided in sequence listings, and thenon-coding strand, used as the template for transcription of a gene orcDNA, can be referred to as encoding the protein or other product ofthat gene or cDNA.

Unless otherwise specified, a “nucleotide sequence encoding an aminoacid sequence” includes all nucleotide sequences that are degenerateversions of each other and that encode the same amino acid sequence.Nucleotide sequences that encode proteins and RNA may include introns.

“Effective amount” or “therapeutically effective amount” are usedinterchangeably herein, and refer to an amount of a compound,formulation, material, or composition, as described herein effective toachieve a particular biological result. Such results may include, butare not limited to, the inhibition of virus infection as determined byany means suitable in the art.

As used herein “endogenous” refers to any material from or producedinside an organism, cell, tissue or system.

As used herein, the term “exogenous” refers to any material introducedfrom or produced outside an organism, cell, tissue or system.

The term “expression” as used herein is defined as the transcriptionand/or translation of a particular nucleotide sequence driven by apromoter.

“Expression vector” refers to a vector comprising a recombinantpolynucleotide comprising expression control sequences operativelylinked to a nucleotide sequence to be expressed. An expression vectorcomprises sufficient cis-acting elements for expression; other elementsfor expression can be supplied by the host cell or in an in vitroexpression system. Expression vectors include all those known in theart, such as cosmids, plasmids (e.g., naked or contained in liposomes),retrotransposons (e.g. piggyback, sleeping beauty), and viruses (e.g.,lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses)that incorporate the recombinant polynucleotide.

“Homologous” as used herein, refers to the subunit sequence identitybetween two polymeric molecules, e.g., between two nucleic acidmolecules, such as, two DNA molecules or two RNA molecules, or betweentwo polypeptide molecules. When a subunit position in both of the twomolecules is occupied by the same monomeric subunit; e.g., if a positionin each of two DNA molecules is occupied by adenine, then they arehomologous at that position. The homology between two sequences is adirect function of the number of matching or homologous positions; e.g.,if half (e.g., five positions in a polymer ten subunits in length) ofthe positions in two sequences are homologous, the two sequences are 50%homologous; if 90% of the positions (e.g., 9 of 10), are matched orhomologous, the two sequences are 90% homologous.

“Identity” as used herein refers to the subunit sequence identitybetween two polymeric molecules particularly between two amino acidmolecules, such as, between two polypeptide molecules. When two aminoacid sequences have the same residues at the same positions; e.g., if aposition in each of two polypeptide molecules is occupied by anArginine, then they are identical at that position. The identity orextent to which two amino acid sequences have the same residues at thesame positions in an alignment is often expressed as a percentage. Theidentity between two amino acid sequences is a direct function of thenumber of matching or identical positions; e.g., if half (e.g., fivepositions in a polymer ten amino acids in length) of the positions intwo sequences are identical, the two sequences are 50% identical; if 90%of the positions (e.g., 9 of 10), are matched or identical, the twoamino acids sequences are 90% identical.

As used herein, an “instructional material” includes a publication, arecording, a diagram, or any other medium of expression which can beused to communicate the usefulness of the compositions and methods ofthe invention. The instructional material of the kit of the inventionmay, for example, be affixed to a container which contains the nucleicacid, peptide, and/or composition of the invention or be shippedtogether with a container which contains the nucleic acid, peptide,and/or composition. Alternatively, the instructional material may beshipped separately from the container with the intention that theinstructional material and the compound be used cooperatively by therecipient.

“Intracellular domain” refers to a portion or region of a molecule thatresides inside a cell.

“Isolated” means altered or removed from the natural state. For example,a nucleic acid or a peptide naturally present in a living animal is not“isolated,” but the same nucleic acid or peptide partially or completelyseparated from the coexisting materials of its natural state is“isolated.” An isolated nucleic acid or protein can exist insubstantially purified form, or can exist in a non-native environmentsuch as, for example, a host cell.

In the context of the present invention, the following abbreviations forthe commonly occurring nucleic acid bases are used. “A” refers toadenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refersto thymidine, and “U” refers to uridine.

Unless otherwise specified, a “nucleotide sequence encoding an aminoacid sequence” includes all nucleotide sequences that are degenerateversions of each other and that encode the same amino acid sequence. Thephrase nucleotide sequence that encodes a protein or an RNA may alsoinclude introns to the extent that the nucleotide sequence encoding theprotein may in some version contain an intron(s).

A “lentivirus” as used herein refers to a genus of the Retroviridaefamily. Lentiviruses are unique among the retroviruses in being able toinfect non-dividing cells; they can deliver a significant amount ofgenetic information into the DNA of the host cell, so they are one ofthe most efficient methods of a gene delivery vector. HIV, SIV, and FIVare all examples of lentiviruses. Vectors derived from lentivirusesoffer the means to achieve significant levels of gene transfer in vivo.

The term “operably linked” refers to functional linkage between aregulatory sequence and a heterologous nucleic acid sequence resultingin expression of the latter. For example, a first nucleic acid sequenceis operably linked with a second nucleic acid sequence when the firstnucleic acid sequence is placed in a functional relationship with thesecond nucleic acid sequence. For instance, a promoter is operablylinked to a coding sequence if the promoter affects the transcription orexpression of the coding sequence. Generally, operably linked DNAsequences are contiguous and, where necessary to join two protein codingregions, in the same reading frame.

“Parenteral” administration of an immunogenic composition includes,e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), orintrasternal injection, or infusion techniques.

The term “polynucleotide” as used herein is defined as a chain ofnucleotides. Furthermore, nucleic acids are polymers of nucleotides.Thus, nucleic acids and polynucleotides as used herein areinterchangeable. One skilled in the art has the general knowledge thatnucleic acids are polynucleotides, which can be hydrolyzed into themonomeric “nucleotides.” The monomeric nucleotides can be hydrolyzedinto nucleosides. As used herein polynucleotides include, but are notlimited to, all nucleic acid sequences which are obtained by any meansavailable in the art, including, without limitation, recombinant means,i.e., the cloning of nucleic acid sequences from a recombinant libraryor a cell genome, using ordinary cloning technology and PCRTM, and thelike, and by synthetic means.

As used herein, the terms “peptide,” “polypeptide,” and “protein” areused interchangeably, and refer to a compound comprised of amino acidresidues covalently linked by peptide bonds. A protein or peptide mustcontain at least two amino acids, and no limitation is placed on themaximum number of amino acids that can comprise a protein's or peptide'ssequence. Polypeptides include any peptide or protein comprising two ormore amino acids joined to each other by peptide bonds. As used herein,the term refers to both short chains, which also commonly are referredto in the art as peptides, oligopeptides and oligomers, for example, andto longer chains, which generally are referred to in the art asproteins, of which there are many types. “Polypeptides” include, forexample, biologically active fragments, substantially homologouspolypeptides, oligopeptides, homodimers, heterodimers, variants ofpolypeptides, modified polypeptides, derivatives, analogs, fusionproteins, among others. The polypeptides include natural peptides,recombinant peptides, synthetic peptides, or a combination thereof.

The term “proinflammatory cytokine” refers to a cytokine or factor thatpromotes inflammation or inflammatory responses. Examples ofproinflammatory cytokines include, but are not limited to, chemokines(CCL, CXCL, CX3CL, XCL), interleukins (such as, IL-1, IL-2, IL-3, IL-5,IL-6, IL-7, IL-9, IL10 and IL-15), interferons (IFNγ), and tumornecrosis factors (TNFα and TNFβ).

The term “promoter” as used herein is defined as a DNA sequencerecognized by the synthetic machinery of the cell, or introducedsynthetic machinery, required to initiate the specific transcription ofa polynucleotide sequence.

As used herein, the term “promoter/regulatory sequence” means a nucleicacid sequence which is required for expression of a gene productoperably linked to the promoter/regulatory sequence. In some instances,this sequence may be the core promoter sequence and in other instances,this sequence may also include an enhancer sequence and other regulatoryelements which are required for expression of the gene product. Thepromoter/regulatory sequence may, for example, be one which expressesthe gene product in a tissue specific manner.

A “constitutive” promoter is a nucleotide sequence which, when operablylinked with a polynucleotide which encodes or specifies a gene product,causes the gene product to be produced in a cell under most or allphysiological conditions of the cell.

An “inducible” promoter is a nucleotide sequence which, when operablylinked with a polynucleotide which encodes or specifies a gene product,causes the gene product to be produced in a cell substantially only whenan inducer which corresponds to the promoter is present in the cell.

A “tissue-specific” promoter is a nucleotide sequence which, whenoperably linked with a polynucleotide encodes or specified by a gene,causes the gene product to be produced in a cell substantially only ifthe cell is a cell of the tissue type corresponding to the promoter.

A “signal transduction pathway” refers to the biochemical relationshipbetween a variety of signal transduction molecules that play a role inthe transmission of a signal from one portion of a cell to anotherportion of a cell. The phrase “cell surface receptor” includes moleculesand complexes of molecules capable of receiving a signal andtransmitting signal across the membrane of a cell.

“Signaling domain” refers to the portion or region of a molecule thatrecruits and interacts with specific proteins in response to anactivating signal.

The term “subject” is intended to include living organisms in which animmune response can be elicited (e.g., mammals).

As used herein, a “substantially purified” cell is a cell that isessentially free of other cell types. A substantially purified cell alsorefers to a cell which has been separated from other cell types withwhich it is normally associated in its naturally occurring state. Insome instances, a population of substantially purified cells refers to ahomogenous population of cells. In other instances, this term referssimply to cells that have been separated from the cells with which theyare naturally associated in their natural state. In some embodiments,the cells are cultured in vitro. In other embodiments, the cells are notcultured in vitro.

The term “therapeutic” as used herein means a treatment and/orprophylaxis. A therapeutic effect is obtained by suppression, remission,or eradication of a disease state.

The term “transfected” or “transformed” or “transduced” as used hereinrefers to a process by which exogenous nucleic acid is transferred orintroduced into the host cell. A “transfected” or “transformed” or“transduced” cell is one which has been transfected, transformed ortransduced with exogenous nucleic acid. The cell includes the primarysubject cell and its progeny.

“Transmembrane domain” refers to a portion or a region of a moleculethat spans a lipid bilayer membrane.

The phrase “under transcriptional control” or “operatively linked” asused herein means that the promoter is in the correct location andorientation in relation to a polynucleotide to control the initiation oftranscription by RNA polymerase and expression of the polynucleotide.

A “vector” is a composition of matter which comprises an isolatednucleic acid and which can be used to deliver the isolated nucleic acidto the interior of a cell. Numerous vectors are known in the artincluding, but not limited to, linear polynucleotides, polynucleotidesassociated with ionic or amphiphilic compounds, plasmids, and viruses.Thus, the term “vector” includes an autonomously replicating plasmid ora virus. The term should also be construed to include non-plasmid andnon-viral compounds which facilitate transfer of nucleic acid intocells, such as, for example, polylysine compounds, liposomes, and thelike. Examples of viral vectors include, but are not limited to,adenoviral vectors, adeno-associated virus vectors, retroviral vectors,lentiviral vectors, and the like.

By the term “specifically binds,” as used herein, is meant an antibody,or a ligand, which recognizes and binds with a cognate binding partner(e.g., a stimulatory and/or costimulatory molecule present on a T cell)protein present in a sample, but which antibody or ligand does notsubstantially recognize or bind other molecules in the sample.

By the term “stimulation,” is meant a primary response induced bybinding of a stimulatory molecule (e.g., a TCR/CD3 complex) with itscognate ligand thereby mediating a signal transduction event, such as,but not limited to, signal transduction via the TCR/CD3 complex.Stimulation can mediate altered expression of certain molecules, such asdownregulation of TGF-β, and/or reorganization of cytoskeletalstructures, and the like.

A “stimulatory molecule,” as the term is used herein, means a moleculeon a T cell that specifically binds with a cognate stimulatory ligandpresent on an antigen presenting cell.

A “stimulatory ligand,” as used herein, means a ligand that when presenton an antigen presenting cell (e.g., an aAPC, a dendritic cell, aB-cell, and the like) can specifically bind with a cognate bindingpartner (referred to herein as a “stimulatory molecule”) on a T cell,thereby mediating a primary response by the T cell, including, but notlimited to, activation, initiation of an immune response, proliferation,and the like. Stimulatory ligands are well-known in the art andencompass, inter alia, an MHC Class I molecule loaded with a peptide, ananti-CD3 antibody, a superagonist anti-CD28 antibody, and a superagonistanti-CD2 antibody.

Ranges: throughout this disclosure, various aspects of the invention canbe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. Thisapplies regardless of the breadth of the range.

Chimeric AutoAntibody Receptor (CAAR)

The present invention is partly based on the discovery that chimericautoantibody receptors can be used to target autoantibodies that causeautoimmune disease. The invention includes compositions comprising atleast one chimeric autoantibody receptor (CAAR) specific for anautoantibody, vectors comprising the same, compositions comprising CAARvectors packaged in viral particles, and recombinant T cells or othereffector cells comprising the CAAR. The invention also includes methodsof making a genetically modified T cell expressing a CAAR (CAART)wherein the expressed CAAR comprises a desmoglein extracellular domain.

The antigens for many of autoantibody-mediated diseases have beendescribed. The present invention includes a technology for treatingautoantibody-mediated diseases. In particular, technologies that targetB cells that ultimately produce the autoantibodies and display theautoantibodies on their cell surfaces, mark these B cells asdisease-specific targets for therapeutic intervention. The inventiontherefore includes a method for efficiently targeting and killing thepathogenic B cells in autoimmune diseases by targeting thedisease-causing B cells using an antigen-specific (e.g., desmoglein 3)chimeric autoantibody receptor (or CAAR). In one embodiment of thepresent invention, only specific anti-Dsg3 autoantibody-expressing Bcells are killed, thus leaving intact the beneficial B cells andantibodies that protect from infection.

The present invention encompasses a recombinant DNA construct comprisingnucleic acid sequences that encode an extracellular domain comprising anautoantigen or a fragment thereof, in one aspect, a human Dsg1, Dsg3 ora fragment thereof, wherein the sequence of the autoantigen or fragmentthereof is operably linked to a nucleic acid sequence of anintracellular signaling domain. The intracellular signaling domain orotherwise the cytoplasmic domain comprises, a costimulatory signalingregion. The costimulatory signaling region refers to a portion of theCAAR comprising the intracellular domain of a costimulatory molecule.Costimulatory molecules are cell surface molecules that are required foran efficient T cell activation.

In one aspect, the invention includes an isolated nucleic acid sequenceencoding a chimeric autoantibody receptor (CAAR), wherein the isolatednucleic acid sequence comprises an extracellular domain comprising anautoantigen or fragment thereof, a nucleic acid sequence of atransmembrane domain, and a nucleic acid sequence of an intracellularsignaling domain.

Autoantigen Moiety

In one exemplary embodiment, a genetically enginereed chimericautoantibody receptor includes the major pemphigus vulgaris autoantigen,desmoglein 3 (Dsg3) or fragments thereof, on the cell surface of Tcells. In this embodiment, the CAAR comprises a propeptide, such as ahuman desmoglein 3 propeptide (amino acids 24-49 of human desmoglein 3):ELRIETKGQYDEEEMTMQQAKRRQKR (SEQ ID NO:2). The human Dsg3 propeptideprevents adhesion of the Dsg3 protein to itself within the syntheticpathway of the cell and is cleaved off by furin or furin-like peptidasesin the late Golgi. In one embodiment, the isolated nucleic acid sequenceencoding the CAAR comprises a nucleic acid sequence of a propeptide ofDsg3. In another embodiment, the propeptide of Dsg3 encodes an aminoacid sequence comprising SEQ ID NO:2. In yet another embodiment, theCAAR comprises a propeptide of Dsg3. In still another embodiment, theCAAR comprises a propeptide of Dsg3 comprising SEQ ID NO:2, such as:

a) human desmoglein 3 extracellular domains 1-5 (amino acids 50-615 ofhuman desmoglein 3, uniprot P32926. The extracellular domains of Dsg3provide the target for autoimmune Dsg3 specific B cells.

(SEQ ID NO: 3) EWVKFAKPCREGEDNSKRNPIAKITSDYQATQKITYRISGVGIDQPPFGIFVVDKNTGDINITAIVDREETPSFLITCRALNAQGLDVEKPLILTVKILDINDNPPVFSQQIFMGEIEENSASNSLVMILNATDADEPNHLNSKIAFKIVSQEPAGTPMFLLSRNTGEVRTLTNSLDREQASSYRLVVSGADKDGEGLSTQCECNIKVKDVNDNFPMFRDSQYSARIEENILSSELLRFQVTDLDEEYTDNWLAVYFFTSGNEGNWFEIQTDPRTNEGILKVVKALDYEQLQSVKLSIAVKNKAEFHQSVISRYRVQSTPVTIQVINVREGIAFRPASKTFTVQKGISSKKLVDYILGTYQAIDEDTNKAASNVKYVMGRNDGGYLMIDSKTAEIKFVKNMNRDSTFIVNKTITAEVLAIDEYTGKTSTGTVYVRVPDFNDNCPTAVLEKDAVCSSSPSVVVSARTLNNRYTGPYTFALEDQPVKLPAVWSITTLNATSALLRAQEQIPPGVYHISLVLTDSQNNRCEMPRSLTLEVCQCDNRGICGTSY PTTSPGTRYGRPHSGR. 

b) same as a), but with only the EC1-2 domains (amino acids 50-268 ofhuman desmoglein 3, P32926). (SEQ ID NO:4).

c) same as a), but with only the EC1-3 domains (amino acids 50-383 ofhuman desmoglein 3, P32926). (SEQ ID NO:5).

d) same as a), but with only the EC1-4 domains (amino acids 50-499 ofhuman desmoglein 3, P32926). (SEQ ID NO:6).

e) same as a), but with only the EC2-3 domains (amino acids 159-383 ofhuman desmoglein 3, P32926). (SEQ ID NO:7).

f) same as a), but with only the EC3-4 domains (amino acids 269-499 ofhuman desmoglein 3, P32926). (SEQ ID NO:8).

g) same as a), but with only the EC4-5 domains (amino acids 386-615 ofhuman desmoglein 3, P32926). (SEQ ID NO:9).

h) same as a), but with only the EC2-4 domains (amino acids 159-499 ofhuman desmoglein 3, P32926). (SEQ ID NO:10).

i) same as a), but with only the EC3-5 domains (amino acids 269-615 ofhuman desmoglein 3, P32926). (SEQ ID NO:11).

k) same as a), but with only the EC2-5 domains (amino acids 159-615 ofhuman desmoglein 3, P32926). (SEQ ID NO:12).

In one embodiment, the nucleic acid sequence of the Dsg3 extracellulardomain encodes an amino acid sequence selected from the group consistingof SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12. Inanother embodiment, the CAAR comprises the Dsg3 extracellular domaincomprising an amino acid sequence selected from the group consisting ofSEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ IDNO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12.

In one embodiment, a nucleic acid sequence has at least 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity or homology toany nucleic acid sequence described herein. In another embodiment, anamino acid sequence has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity or homology to any amino acid sequencedescribed herein.

In another aspect, the constructs described herein comprise anextracellular domain comprising an autoantigen or fragment thereof. Inone embodiment, the autoantigen is selected from the group consisting ofDsg1, Dsg3, and a fragment thereof.

In one embodiment, the CAAR of the invention comprises an autoantibodybinding domain otherwise referred to as an autoantigen or a fragmentthereof. The choice of autoantigen for use in the present inventiondepends upon the type of autoantibody being targeted. For example, theautoantigen may be chosen because it recognizes an autoantibody on atarget cell, such as a B cell, associated with a particular diseasestate, e.g. an autoimmune disease.

In some instances, it is beneficial that the autoantibody binding domainis derived from the same species in which the CAAR will ultimately beused. For example, for use in humans, it may be beneficial that theautoantibody binding domain of the CAAR comprises an autoantigen thatbinds an autoantibody or a fragment thereof. Thus, in one embodiment,the autoantibody binding domain portion comprises an epitope of theautoantigen that binds the autoantibody. The epitope is the part of theautoantigen that is specifically recognized by the autoantibody.

Transmembrane Domain

In one embodiment, the CAAR comprises a transmembrane domain, such as,but not limited to, a human T cell surface glycoprotein CD8 alpha chainhinge and/or transmembrane domain (amino acids 136-203 of the humanhuman T cell surface glycoprotein CD8 alpha chain).KPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPL AGTCGVLLLSLVIT(SEQ ID NO:13). The human CD8 chain hinge and/or transmembrane domainprovides cell surface presentation of the chimeric autoantibodyreceptor.

With respect to the transmembrane domain, in various embodiments, theCAAR comprises a transmembrane domain that is fused to the extracellulardomain of the CAAR. In one embodiment, the CAAR comprises atransmembrane domain that naturally is associated with one of thedomains in the CAAR. In some instances, the transmembrane domain is beselected or modified by amino acid substitution to avoid binding to thetransmembrane domains of the same or different surface membrane proteinsin order to minimize interactions with other members of the receptorcomplex.

The transmembrane domain may be derived either from a natural or from asynthetic source. When the source is natural, the domain may be derivedfrom any membrane-bound or transmembrane protein. In one embodiment, thetransmembrane domain may be synthetic, in which case it will comprisepredominantly hydrophobic residues such as leucine and valine. In oneaspect a triplet of phenylalanine, tryptophan and valine will be foundat each end of a synthetic transmembrane domain. Optionally, a shortoligo- or polypeptide linker, between 2 and 10 amino acids in length mayform the linkage between the transmembrane domain and the cytoplasmicsignaling domain of the CAAR. A glycine-serine doublet provides aparticularly suitable linker.

In some instances, a variety of human hinges can be employed as wellincluding the human Ig (immunoglobulin) hinge.

Examples of the hinge and/or transmembrane domain include, but are notlimited to, a hinge and/or transmembrane domain of an alpha, beta orzeta chain of a T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8,CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIR,OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR,CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, IL2Rbeta, IL2R gamma, IL7R α, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6,VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM,CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2,DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1,CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A,Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162),LTBR, PAG/Cbp, NKp44, NKp30, NKp46, NKG2D, and/or NKG2C.

In one embodiment, the nucleic acid sequence of the transmembrane domainencodes a CD8 alpha chain hinge and/or transmembrane domain. In anotherembodiment, the nucleic acid sequence of the CD8 alpha chain hingeand/or transmembrane domain encodes an amino acid sequence comprisingSEQ ID NO:13.

In yet another embodiment, the transmembrane domain comprises a CD8alpha chain hinge and/or transmembrane domain.

Cytoplasmic Domain

The cytoplasmic domain or otherwise the intracellular signaling domainof the CAAR of the invention, is responsible for activation of at leastone of the normal effector functions of the immune cell in which theCAAR has been placed in.

The term “effector function” refers to a specialized function of a cell.

Effector function of a T cell, for example, may be cytolytic activity orhelper activity including the secretion of cytokines. Thus the term“intracellular signaling domain” refers to the portion of a proteinwhich transduces the effector function signal and directs the cell toperform a specialized function. While the entire intracellular signalingdomain can be employed, in many cases it is not necessary to use theentire domain. To the extent that a truncated portion of theintracellular signaling domain is used, such truncated portion may beused in place of the intact domain as long as it transduces the effectorfunction signal.

The term “intracellular signaling domain” is thus meant to include anytruncated portion of the intracellular domain sufficient to transducethe effector function signal.

Examples of intracellular signaling domains for use in the CAAR of theinvention include, but are not limited to, the cytoplasmic portion ofthe T cell receptor (TCR) and co-receptors that act in concert toinitiate signal transduction following antigen receptor engagement, aswell as any derivative or variant of these elements and any syntheticsequence that has the same functional capability.

It is well recognized that signals generated through the TCR alone areinsufficient for full activation of the T cell and that a secondary orco-stimulatory signal is also required. Thus, T cell activation can besaid to be mediated by two distinct classes of cytoplasmic signalingsequence: those that initiate antigen-dependent primary activationthrough the TCR (primary cytoplasmic signaling sequences) and those thatact in an antigen-independent manner to provide a secondary orco-stimulatory signal (secondary cytoplasmic signaling sequences).

Primary cytoplasmic signaling sequences regulate primary activation ofthe TCR complex either in a stimulatory manner or in an inhibitorymanner. Primary cytoplasmic signaling sequences that act in astimulatory manner may contain signaling motifs which are known asimmunoreceptor tyrosine-based activation motifs or ITAMs.

Examples of the intracellular signaling domain includes a fragment ordomain from one or more molecules or receptors including, but are notlimited to, CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, CD86, commonFcR gamma, FcR beta (Fc Epsilon R1b), CD79a, CD79b, Fcgamma RIIa, DAP10,DAP12, T cell receptor (TCR), CD27, CD28, 4-1BB (CD137), OX40, CD30,CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2,CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83,CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD127,CD160, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha,ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD,CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c,ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE/RANKL, DNAM1(CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9(CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A,Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162),LTBR, LAT, GADS, SLP-76, PAG/Cbp, NKp44, NKp30, NKp46, NKG2D, otherco-stimulatory molecules described herein, any derivative, variant, orfragment thereof, any synthetic sequence of a co-stimulatory moleculethat has the same functional capability, and any combination thereof.

In a preferred embodiment, the intracellular signaling domain of theCAAR comprises the CD3-zeta signaling domain by itself or in combinationwith any other desired cytoplasmic domain(s) useful in the context ofthe CAAR of the invention. For example, the intracellular signalingdomain of the CAAR can comprise a CD3 zeta chain portion and acostimulatory signaling region. The costimulatory signaling regionrefers to a portion of the CAAR comprising the intracellular domain of acostimulatory molecule. A costimulatory molecule is a cell surfacemolecule other than an antigen receptor or its ligands that is requiredfor an efficient response of lymphocytes to an antigen.

In yet another embodiment, the intracellular signaling domain encodes aCD137 intracellular domain. In still another embodiment, the CD137intracellular domain comprisess SEQ ID NO:15, such as a human T-cellsurface glycoprotein CD3 zeta chain isoform 3 intracellular domain(human CD247, amino acids 52-163) RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLS TATKDT YDALHMQALPPR (SEQ IDNO:16). The human intracellular CD3 zeta domain provides stimulatoryintracellular signaling upon binding to the extracellular autoantigen,such as Dsg1, Dsg3, or a fragment thereof, without HLA restriction.

In another embodiment, the nucleic acid sequence of the intracellularsignaling domain comprises a nucleic acid sequence encoding a CD3 zetasignaling domain. In another embodiment, the nucleic acid sequence ofthe CD3 zeta signaling domain encodes an amino acid sequence comprisingSEQ ID NO:16.

In yet another embodiment, the intracellular signaling domain comprisesa CD3 zeta signaling domain. In still another embodiment, the CD3 zetasignaling domain comprises SEQ ID NO:16.

Other Domains

In another embodiment, the CAAR and the nucleic acid encoding the CAARcomprise a human T cell surface glycoprotein CD8 alpha chain signalpeptide (amino acids 1-21 of the T-cell surface glycoprotein CD8 alphachain): MALPVTALLLPLALLLHAARP (SEQ ID NO:1). The human CD8 alpha signalpeptide is responsible for the translocation of the receptor to the Tcell surface. In one embodiment, the isolated nucleic acid sequenceencoding the CAAR comprises a nucleic acid sequence of a CD8 alpha chainsignal peptide. In another embodiment, the CD8 alpha chain signalpeptide encodes an amino acid sequence comprising SEQ ID NO:1. In yetanother embodiment, the CAAR comprises a CD8 alpha chain signal peptide.

In still another embodiment, the transmembrane domain comprises a CD8alpha chain hinge and transmembrane domain comprising SEQ ID NO:13, suchas the hinge region mentioned in a) replaced with a peptide linkerconsisting of the amino acids: SGGGGSGGGGS SG (SEQ ID NO:14) between theEC domains of desmoglein 3 and the CD8 transmembrane domain.

In one embodiment, the isolated nucleic acid sequence encoding the CAARcomprises a nucleic acid sequence of a peptide linker. In anotherembodiment, the nucleic acid sequence of peptide linker encodes an aminoacid sequence comprising SEQ ID NO:14. In another embodiment, thecytoplasmic signaling sequences within the intracellular signalingdomain of the CAAR can be linked to each other in a random or specifiedorder. Optionally, a short oligo- or polypeptide linker, for example,between 2 and 10 amino acids in length may form the linkage. Aglycine-serine doublet is a particularly suitable linker.

In yet another embodiment, the CAAR comprises a peptide linker. In stillanother embodiment, the peptide linker comprises SEQ ID NO:14, such as ahuman tumor necrosis factor receptor superfamily member 9 (also known asCD137 or 4-1BB ligand receptor) intracellular domain (amino acids214-255 of the human tumor necrosis factor receptor superfamily member9):

(SEQ ID NO: 15) KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL.The human intracellular CD137 domain provides co-stimulatoryintracellular signaling upon binding to the extracellular autoantigen,such as Dsg1, Dsg3, or a fragment thereof, without the need of itsoriginal ligand.

Any domains and/or fragments of the CAAR, vector, and the promoter maybe amplified by PCR or any other means known in the art.

Vector Comprising the CAAR

All vectors described herein comprising different parts of anextracellular portion of human Desmoglein 3 should be construed to beequally compatible with use of human Desmoglein 1 extracellular portion.As such, use of the vectors described herein is exemplified by use ofDesmoglein 3, but should be construed to be equally disclosed withrespect to use of human Desmoglein 1.

For proof of concept as to specificity and functionality, a 3^(rd)generation self-inactivating lentiviral vector plasmid is useful, inwhich the expression is regulated under the human elongation factor 1alpha promoter (e.g., pRRLSIN.cPPT.EF1a.Dsg3CAAR.WPRE). This results instable (permanent) expression in the host T cell. As an alternativeapproach, the encoding mRNA can be electroporated into the host cell,which would achieve the same therapeutic effect as the virallytransduced T cells, but would not be permanent, since the mRNA woulddilute out with cell division.

In one aspect, the invention includes a vector comprising an isolatednucleic acid sequence encoding a chimeric autoantibody receptor (CAAR),wherein the isolated nucleic acid sequence comprises a human nucleicacid sequence of an extracellular domain comprising an autoantigen orfragment thereof, a nucleic acid sequence of a transmembrane domain, anda nucleic acid sequence of an intracellular signaling domain. In oneembodiment, the vector comprises any of the isolated nucleic acidsequences encoding the CAAR as described herein. In another embodiment,the vector comprises a plasmid vector, viral vector, retrotransposon(e.g. piggyback, sleeping beauty), site directed insertion vector (e.g.CRISPR, zn finger nucleases, TALEN), or suicide expression vector, orother known vector in the art.

All constructs mentioned above comprising different autoantigens andfragments thereof are capable of use with 3rd generation lentiviralvector plasmids, other viral vectors, or RNA approved for use in humancells. In one embodiment, the vector is a viral vector, such as alentiviral vector. In another embodiment, the vector is a RNA vector.

The production of the CAAR can be verified by sequencing. Expression ofthe full length CAAR protein may be verified using immunoblot,immunohistochemistry, flow cytometry or other technology well known andavailable in the art.

The present invention also provides a vector in which DNA encoding theCAAR of the present invention is inserted. Vectors, including thosederived from retroviruses such as lentivirus, are suitable tools toachieve long-term gene transfer since they allow long-term, stableintegration of a transgene and its propagation in daughter cells.Lentiviral vectors have the added advantage over vectors derived fromonco-retroviruses, such as murine leukemia viruses, in that they cantransduce non-proliferating cells, such as hepatocytes. They also havethe added advantage of resulting in low immunogenicity in the subjectinto which they are introduced.

In brief summary, the expression of natural or synthetic nucleic acidsencoding CAARs is typically achieved by operably linking a nucleic acidencoding the CAAR polypeptide or portions thereof to a promoter, andincorporating the construct into an expression vector. The vector is onegenerally capable of replication in a mammalian cell, and/or alsocapable of integration into the cellular genome of the mammal. Typicalvectors contain transcription and translation terminators, initiationsequences, and promoters useful for regulation of the expression of thedesired nucleic acid sequence.

The nucleic acid can be cloned into any number of different types ofvectors. For example, the nucleic acid can be cloned into a vectorincluding, but not limited to a plasmid, a phagemid, a phage derivative,an animal virus, and a cosmid. Vectors of particular interest includeexpression vectors, replication vectors, probe generation vectors, andsequencing vectors.

The expression vector may be provided to a cell in the form of a viralvector. Viral vector technology is well known in the art and isdescribed, for example, in Sambrook et al., 2012, MOLECULAR CLONING: ALABORATORY MANUAL, volumes 1-4, Cold Spring Harbor Press, NY), and inother virology and molecular biology manuals. Viruses, which are usefulas vectors include, but are not limited to, retroviruses, adenoviruses,adeno-associated viruses, herpes viruses, and lentiviruses. In general,a suitable vector contains an origin of replication functional in atleast one organism, a promoter sequence, convenient restrictionendonuclease sites, and one or more selectable markers, (e.g., WO01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193).

Additional promoter elements, e.g., enhancers, regulate the frequency oftranscriptional initiation. Typically, these are located in the region30-110 bp upstream of the start site, although a number of promotershave recently been shown to contain functional elements downstream ofthe start site as well. The spacing between promoter elements frequentlyis flexible, so that promoter function is preserved when elements areinverted or moved relative to one another. In the thymidine kinase (tk)promoter, the spacing between promoter elements can be increased to 50bp apart before activity begins to decline. Depending on the promoter,it appears that individual elements can function either cooperatively orindependently to activate transcription.

An example of a promoter is the immediate early cytomegalovirus (CMV)promoter sequence. This promoter sequence is a strong constitutivepromoter sequence capable of driving high levels of expression of anypolynucleotide sequence operatively linked thereto. However, otherconstitutive promoter sequences may also be used, including, but notlimited to the simian virus 40 (SV40) early promoter, mouse mammarytumor virus (MMTV), human immunodeficiency virus (HIV) long terminalrepeat (LTR) promoter, MoMuLV promoter, an avian leukemia viruspromoter, an Epstein-Barr virus immediate early promoter, a Rous sarcomavirus promoter, the elongation factor-1α promoter, as well as human genepromoters such as, but not limited to, the actin promoter, the myosinpromoter, the hemoglobin promoter, and the creatine kinase promoter.Further, the invention should not be limited to the use of constitutivepromoters. Inducible promoters are also contemplated as part of theinvention. The use of an inducible promoter provides a molecular switchcapable of turning on expression of the polynucleotide sequence which itis operatively linked when such expression is desired, or turning offthe expression when expression is not desired. Examples of induciblepromoters include, but are not limited to a metallothionine promoter, aglucocorticoid promoter, a progesterone promoter, and a tetracyclinepromoter.

In order to assess the expression of a CAAR polypeptide or portionsthereof, the expression vector to be introduced into a cell can alsocontain either a selectable marker gene or a reporter gene or both tofacilitate identification and selection of expressing cells from thepopulation of cells sought to be transfected or infected through viralvectors. In other aspects, the selectable marker may be carried on aseparate piece of DNA and used in a co-transfection procedure. Bothselectable markers and reporter genes may be flanked with appropriateregulatory sequences to enable expression in the host cells. Usefulselectable markers include, for example, antibiotic-resistance genes,such as neo and the like.

Reporter genes are used for identifying potentially transfected cellsand for evaluating the functionality of regulatory sequences. Ingeneral, a reporter gene is a gene that is not present in or expressedby the recipient organism or tissue and that encodes a polypeptide whoseexpression is manifested by some easily detectable property, e.g.,enzymatic activity. Expression of the reporter gene is assessed at asuitable time after the DNA has been introduced into the recipientcells. Suitable reporter genes may include genes encoding luciferase,beta-galactosidase, chloramphenicol acetyl transferase, secretedalkaline phosphatase, or the green fluorescent protein gene (e.g.,Ui-Tei et al., 2000 FEBS Letters 479: 79-82). Suitable expressionsystems are well known and may be prepared using known techniques orobtained commercially. In general, the construct with the minimal 5′flanking region showing the highest level of expression of reporter geneis identified as the promoter. Such promoter regions may be linked to areporter gene and used to evaluate agents for the ability to modulatepromoter-driven transcription.

Methods of introducing and expressing genes into a cell are known in theart. In the context of an expression vector, the vector can be readilyintroduced into a host cell, e.g., mammalian, bacterial, yeast, orinsect cell by any method in the art. For example, the expression vectorcan be transferred into a host cell by physical, chemical, or biologicalmeans.

Physical methods for introducing a polynucleotide into a host cellinclude calcium phosphate precipitation, lipofection, particlebombardment, microinjection, electroporation, and the like. Methods forproducing cells comprising vectors and/or exogenous nucleic acids arewell-known in the art. See, for example, Sambrook et al., 2012,MOLECULAR CLONING: A LABORATORY MANUAL, volumes 1-4, Cold Spring HarborPress, NY).

Biological methods for introducing a polynucleotide of interest into ahost cell include the use of DNA and RNA vectors. RNA vectors includevectors having a RNA promoter and/other relevant domains for productionof a RNA transcript. Viral vectors, and especially retroviral vectors,have become the most widely used method for inserting genes intomammalian, e.g., human cells. Other viral vectors may be derived fromlentivirus, poxviruses, herpes simplex virus, adenoviruses andadeno-associated viruses, and the like. See, for example, U.S. Pat. Nos.5,350,674 and 5,585,362.

Chemical means for introducing a polynucleotide into a host cell includecolloidal dispersion systems, such as macromolecule complexes,nanocapsules, microspheres, beads, and lipid-based systems includingoil-in-water emulsions, micelles, mixed micelles, and liposomes. Anexemplary colloidal system for use as a delivery vehicle in vitro and invivo is a liposome (e.g., an artificial membrane vesicle).

In the case where a non-viral delivery system is utilized, an exemplarydelivery vehicle is a liposome. The use of lipid formulations iscontemplated for the introduction of the nucleic acids into a host cell(in vitro, ex vivo or in vivo). In another aspect, the nucleic acid maybe associated with a lipid. The nucleic acid associated with a lipid maybe encapsulated in the aqueous interior of a liposome, interspersedwithin the lipid bilayer of a liposome, attached to a liposome via alinking molecule that is associated with both the liposome and theoligonucleotide, entrapped in a liposome, complexed with a liposome,dispersed in a solution containing a lipid, mixed with a lipid, combinedwith a lipid, contained as a suspension in a lipid, contained orcomplexed with a micelle, or otherwise associated with a lipid. Lipid,lipid/DNA or lipid/expression vector associated compositions are notlimited to any particular structure in solution. For example, they maybe present in a bilayer structure, as micelles, or with a “collapsed”structure. They may also simply be interspersed in a solution, possiblyforming aggregates that are not uniform in size or shape. Lipids arefatty substances which may be naturally occurring or synthetic lipids.For example, lipids include the fatty droplets that naturally occur inthe cytoplasm as well as the class of compounds which contain long-chainaliphatic hydrocarbons and their derivatives, such as fatty acids,alcohols, amines, amino alcohols, and aldehydes.

Lipids suitable for use can be obtained from commercial sources. Forexample, dimyristyl phosphatidylcholine (“DMPC”) can be obtained fromSigma, St. Louis, Mo.; dicetyl phosphate (“DCP”) can be obtained from K& K Laboratories (Plainview, N.Y.); cholesterol (“Choi”) can be obtainedfrom Calbiochem-Behring; dimyristyl phosphatidylglycerol (“DMPG”) andother lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham,Ala.). Stock solutions of lipids in chloroform or chloroform/methanolcan be stored at about −20° C. Chloroform is used as the only solventsince it is more readily evaporated than methanol. “Liposome” is ageneric term encompassing a variety of single and multilamellar lipidvehicles formed by the generation of enclosed lipid bilayers oraggregates. Liposomes can be characterized as having vesicularstructures with a phospholipid bilayer membrane and an inner aqueousmedium. Multilamellar liposomes have multiple lipid layers separated byaqueous medium. They form spontaneously when phospholipids are suspendedin an excess of aqueous solution. The lipid components undergoself-rearrangement before the formation of closed structures and entrapwater and dissolved solutes between the lipid bilayers (Ghosh et al.,1991 Glycobiology 5: 505-10). However, compositions that have differentstructures in solution than the normal vesicular structure are alsoencompassed. For example, the lipids may assume a micellar structure ormerely exist as nonuniform aggregates of lipid molecules. Alsocontemplated are lipofectamine-nucleic acid complexes.

Cells Comprising the CAAR

In another aspect, the invention includes a genetically modified cell,such as a helper T cell, a cytotoxic T cell, a memory T cell, regulatoryT cell, gamma delta T cell, a natural killer cell, cytokine inducedkiller cell, a cell line thereof, and other effector cell, comprises achimeric autoantibody receptor (CAAR), wherein the CAAR comprises anextracellular domain comprising an autoantigen or fragment thereof, atransmembrane domain, and an intracellular signaling domain. In oneembodiment, the genetically modified cell comprises the CAAR describedherein.

In another embodiment, the cell expresses the CAAR. In this embodiment,the cell has high affinity for autoantibodies expressed on B cells. As aresult, the cell can induce killing of B cells expressing theautoantibodies. In yet another embodiment, the cell has low affinity forantibodies bound to a Fc receptor.

In another embodiment, the genetically modified cell is a T cell. Inthis embodiment, the T cell expresses a Dsg3 CAAR. In this embodiment,the autoantigen comprises Dsg3 or a fragment thereof and the T cell hashigh affinity for Dsg3 autoantibodies expressed on B cells. As a result,the T cell can induce killing of B cells expressing Dsg3 autoantibodies.In yet another embodiment, the autoantigen comprises Dsg3 and the T cellhas low affinity for Dsg3 antibodies bound to a Fc receptor.

It is also useful for the T cell to have limited toxicity toward healthycells and specificity to cells expressing autoantibodies. Suchspecificity prevents or reduces off-target toxicity that is prevalent incurrent therapies that are not specific for autoantibodies. In oneembodiment the T cell has limited toxicity toward healthy cells.

The invention includes T cells, such as primary cells, expanded T cellsderived from primary T cells, T cells derived from stem cellsdifferentiated in vitro, T cell lines such as Jurkat cells, othersources of T cells, combinations thereof, and other effector cells. Forexample, a transduced Jurkat cell line with a NFAT response elementfollowed by GFP can be used to detect and isolate Dsg3 specific B cellsand to clone the Dsg3 specific antibody repertoire in a comprehensiveand unbiased fashion. The interacting B and Jurkat cells can be detectedas GFP positive doublets and sorted by flow cytometry. Expressioncloning of the B cell receptor encoding genes will provide furtherinformation on how autoimmunity and autoantibodies in pemphigus, such aspemphigus vulgaris, paraneoplastic pemphigus or pemphigus foliaceus, andother autoimmune diseases develop.

The functional ability of CAARs to bind to autoantibodies and sera, forexample, but not limited to, pemphigus vulgaris sera, has been assessedin a Jurkat reporter cell line, which depends on activation of the CAARby binding to autoantibody (in response to which the activated cellsfluoresce green due to an NFAT-GFP reporter construct containedtherein). Such methods are useful and reliable qualitative measures forfunctional binding ability. The proper processing of the autoantigen onthe cell surface is also important and can be measured using monoclonalantibodies. For example, as described herein, a serial dilution ofanti-Dsg3 hybridoma cells (AK23) showed a dose dependent response of thetransduced Jurkat cells to the Dsg3 autoantibody displaying hybridomaand no activation by non-Dsg3 specific or healthy primary human B cells.Furthermore, primary T cells transduced with the CAAR demonstratespecific killing of AK23. Furthermore, truncations of Dsg3 based onmajor disease epitopes are also useful and included herein. Truncatedversions using a smaller hinge region are also useful. With regard tosafety, preventing or reducing possible hemophilic and heterophilicinteractions and activation (e.g. Dsg3-Dsg3) between the transducedcells or toward keratinocytes is preferred.

Further assessment of efficacy and safety of the CAAR can be performed,for example, as follows:

Constructs can be transiently transfected into human cells, such as293T/17. The surface expression can be detected with monoclonalantibodies (either IgG or ScFv) specific for the abovementionedextracellular domains 1,2,3,4,5, the linker between the domains, orother structure included in the CAAR. Binding can be verfied withspecific secondary antibodies and quantified by flow cytometry.

Production of membrane expressed constructs of human anti-desmoglein 3antibodies of the IgM and IgG1 isotype is described hererin, which areexpressed in MHCI negative K562 cells (ATCC CCL-243). These cells canserve as target cells for testing the different Dsg3-CAARs.

The above mentioned the CAAR constructs are compatible with VSV-Gpseudotyped HIV-1 derived lentiviral particles and can be permanentlyexpressed in primary human T cells from healthy donors using lentiviraltransduction. Killing efficacy can be determined in a chromium basedcell lysis assay or any similar assay known in the art.

Additional target cell lines can be produced as needed by expression ofhuman monoclonal antibodies on the surface of K562 cells.

A similar approach as above could be applied to desmoglein 1, againstwhich antibodies are found in mucocutaneous forms of pemphigus vulgarisor pemphigus foliaceus. A similar approach can also be applied to theNC16A domain of BP180 (Type XVII collagen), which is the primaryantigenic target of autoantibodies in bullous pemphigoid; the NC1 andNC2 domains of type VII collagen, which is targeted by autoantibodies inepidermolysis bullosa acquisita; or tissue tranglutaminase/gliadinpeptide/epidermal transglutaminase, which are targeted by autoantibodycomplexes in celiac disease and dermatitis herpetiformis.

Autoimmune Diseases

The present invention also provides methods for preventing, treatingand/or managing a disorder associated with autoantibody-expressing cells(e.g., an autoimmune disease). The methods comprise administering to asubject in need thereof a CAART cell of the invention that binds to theautoantibody-expressing cell. In one aspect, the subject is a human.Non-limiting examples of disorders associated withautoantibody-expressing cells include autoimmune disorders (such aspemphigus vulgaris, paraneoplastic pemphigus or pemphigus foliaceus).

The present invention also provides methods for preventing, treatingand/or managing an autoimmune disease associated withautoantibody-expressing cells. The methods comprise administering to asubject in need a CAART cell of the invention that binds to theautoantibody-expressing cell. In one embodiment, the subject undergoesplasmapheresis or another clinical treatment to remove or decreaseantibodies in the subject's serum. The method to remove or decreaseserum antibodies, such as autoantibodies, may include chemical or othermethods known in the art. The treatment method may be specific to theautoantibody or generalized for any antibody. In one embodiment, thesubject is a human. Non-limiting examples of diseases associated withautoantibody-expressing cells include desmoglein 3 autoantibodies, andthe like.

In the methods of treatment, T cells isolated from a subject can bemodified to express the appropriate CAAR, expanded ex vivo and thenreinfused into the subject. The modified T cells recognize target cells,such as Dsg3 specific B cells, and become activated, resulting inkilling of the autoimmune target cells.

Relapse may also occur in patients with an autoimmune disease, forexample in pemphigus patients. In patients treated with rituximab, therelapse may be mediated by persistence of the same autoantibody B cellclones, whereas remission is associated with disappearance of theseclones. By infusing CAART cells, the autoimmune cells are depleted toinduce long-term remission, possibly due to the longevity of the CAARTcells and/or autoantigen-reactive clones do not re-appear (i.e. inpemphigus vulgaris, paraneoplastic pemphigus or pemphigus foliaceus, thebreak in tolerance is a one-time mistake).

To monitor CAAR-expressing cells in vitro, in situ, or in vivo, CAARcells can further express a detectable marker. When the CAAR binds thetarget, the detectable marker is activated and expressed, which can bedetected by assays known in the art, such as flow cytometry. In oneembodiment, the Dsg3-CAAR includes a NFAT response element and adetectable marker, such as a green fluorescent protein (GFP), to detectand quantify Dsg3 CAAR expressing cells.

Sources of T Cells

Prior to expansion and genetic modification, T cells are obtained from asubject. Examples of subjects include humans, dogs, cats, mice, rats,and transgenic species thereof. T cells can be obtained from a number ofsources, including skin, peripheral blood mononuclear cells, bonemarrow, lymph node tissue, cord blood, thymus tissue, tissue from a siteof infection, ascites, pleural effusion, spleen tissue, and tumors. Incertain embodiments of the present invention, any number of T cell linesavailable in the art, may be used. In certain embodiments of the presentinvention, T cells can be obtained from a unit of blood collected from asubject using any number of techniques known to the skilled artisan,such as Ficoll™ separation. In one preferred embodiment, cells from thecirculating blood of an individual are obtained by apheresis. Theapheresis product typically contains lymphocytes, including T cells,monocytes, granulocytes, B cells, other nucleated white blood cells, redblood cells, and platelets. In one embodiment, the cells collected byapheresis may be washed to remove the plasma fraction and to place thecells in an appropriate buffer or media for subsequent processing steps.In one embodiment of the invention, the cells are washed with phosphatebuffered saline (PBS). In an alternative embodiment, the wash solutionlacks calcium and may lack magnesium or may lack many if not alldivalent cations. Again, surprisingly, initial activation steps in theabsence of calcium lead to magnified activation. As those of ordinaryskill in the art would readily appreciate a washing step may beaccomplished by methods known to those in the art, such as by using asemi-automated “flow-through” centrifuge (for example, the Cobe 2991cell processor, the Baxter CytoMate, or the Haemonetics Cell Saver 5)according to the manufacturer's instructions. After washing, the cellsmay be resuspended in a variety of biocompatible buffers, such as, forexample, Ca-free, Mg-free PBS, PlasmaLyte A, or other saline solutionwith or without buffer. Alternatively, the undesirable components of theapheresis sample may be removed and the cells directly resuspended inculture media.

In another embodiment, T cells are isolated from peripheral bloodlymphocytes by lysing the red blood cells and depleting the monocytes,for example, by centrifugation through a PERCOLL™ gradient or bycounterflow centrifugal elutriation. A specific subpopulation of Tcells, such as CD3⁺, CD28⁺, CD4⁺, CD8⁺, CD45RA⁺, and CD45RO⁻T cells, canbe further isolated by positive or negative selection techniques. Forexample, in one embodiment, T cells are isolated by incubation withanti-CD3/anti-CD28 (i.e., 3×28)-conjugated beads, such as DYNABEADS®M-450 CD3/CD28 T, for a time period sufficient for positive selection ofthe desired T cells. In one embodiment, the time period is about 30minutes. In a further embodiment, the time period ranges from 30 minutesto 36 hours or longer and all integer values there between. In a furtherembodiment, the time period is at least 1, 2, 3, 4, 5, or 6 hours. Inyet another preferred embodiment, the time period is 10 to 24 hours. Inone preferred embodiment, the incubation time period is 24 hours. Forisolation of T cells from patients with leukemia, use of longerincubation times, such as 24 hours, can increase cell yield. Longerincubation times may be used to isolate T cells in any situation wherethere are few T cells as compared to other cell types, such in isolatingtumor infiltrating lymphocytes (TIL) from tumor tissue or fromimmunocompromised individuals. Further, use of longer incubation timescan increase the efficiency of capture of CD8+ T cells. Thus, by simplyshortening or lengthening the time T cells are allowed to bind to theCD3/CD28 beads and/or by increasing or decreasing the ratio of beads toT cells (as described further herein), subpopulations of T cells can bepreferentially selected for or against at culture initiation or at othertime points during the process. Additionally, by increasing ordecreasing the ratio of anti-CD3 and/or anti-CD28 antibodies on thebeads or other surface, subpopulations of T cells can be preferentiallyselected for or against at culture initiation or at other desired timepoints. The skilled artisan would recognize that multiple rounds ofselection can also be used in the context of this invention. In certainembodiments, it may be desirable to perform the selection procedure anduse the “unselected” cells in the activation and expansion process.“Unselected” cells can also be subjected to further rounds of selection.

Enrichment of a T cell population by negative selection can beaccomplished with a combination of antibodies directed to surfacemarkers unique to the negatively selected cells. One method is cellsorting and/or selection via negative magnetic immunoadherence or flowcytometry that uses a cocktail of monoclonal antibodies directed to cellsurface markers present on the cells negatively selected. For example,to enrich for CD4⁺ cells by negative selection, a monoclonal antibodycocktail typically includes antibodies to CD14, CD20, CD11b, CD16,HLA-DR, and CD8. In certain embodiments, it may be desirable to enrichfor or positively select for regulatory T cells which typically expressCD4⁺, CD25⁺, CD62L^(hi), GITR⁺, and FoxP3⁺. Alternatively, in certainembodiments, T regulatory cells are depleted by anti-C25 conjugatedbeads or other similar method of selection.

For isolation of a desired population of cells by positive or negativeselection, the concentration of cells and surface (e.g., particles suchas beads) can be varied. In certain embodiments, it may be desirable tosignificantly decrease the volume in which beads and cells are mixedtogether (i.e., increase the concentration of cells), to ensure maximumcontact of cells and beads. For example, in one embodiment, aconcentration of 2 billion cells/ml is used. In one embodiment, aconcentration of 1 billion cells/ml is used. In a further embodiment,greater than 100 million cells/ml is used. In a further embodiment, aconcentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 millioncells/ml is used. In yet another embodiment, a concentration of cellsfrom 75, 80, 85, 90, 95, or 100 million cells/ml is used. In furtherembodiments, concentrations of 125 or 150 million cells/ml can be used.Using high concentrations can result in increased cell yield, cellactivation, and cell expansion. Further, use of high cell concentrationsallows more efficient capture of cells that may weakly express targetantigens of interest, such as CD28-negative T cells, or from sampleswhere there are many tumor cells present (i.e., leukemic blood, tumortissue, etc.). Such populations of cells may have therapeutic value andwould be desirable to obtain. For example, using high concentration ofcells allows more efficient selection of CD8⁺ T cells that normally haveweaker CD28 expression.

In a related embodiment, it may be desirable to use lower concentrationsof cells. By significantly diluting the mixture of T cells and surface(e.g., particles such as beads), interactions between the particles andcells is minimized. This selects for cells that express high amounts ofdesired antigens to be bound to the particles. For example, CD4⁺ T cellsexpress higher levels of CD28 and are more efficiently captured thanCD8⁺ T cells in dilute concentrations. In one embodiment, theconcentration of cells used is 5×10⁶/ml. In other embodiments, theconcentration used can be from about 1×10⁵/ml to 1×10⁶/ml, and anyinteger value in between.

In other embodiments, the cells may be incubated on a rotator forvarying lengths of time at varying speeds at either 2-10° C. or at roomtemperature.

T cells for stimulation can also be frozen after a washing step. Wishingnot to be bound by theory, the freeze and subsequent thaw step providesa more uniform product by removing granulocytes and to some extentmonocytes in the cell population. After the washing step that removesplasma and platelets, the cells may be suspended in a freezing solution.While many freezing solutions and parameters are known in the art andwill be useful in this context, one method involves using PBS containing20% DMSO and 8% human serum albumin, or culture media containing 10%Dextran 40 and 5% Dextrose, 20% Human Serum Albumin and 7.5% DMSO, or31.25% Plasmalyte-A, 31.25% Dextrose 5%, 0.45% NaCl, 10% Dextran 40 and5% Dextrose, 20% Human Serum Albumin, and 7.5% DMSO or other suitablecell freezing media containing for example, Hespan and PlasmaLyte A, thecells then are frozen to −80° C. at a rate of 1° per minute and storedin the vapor phase of a liquid nitrogen storage tank. Other methods ofcontrolled freezing may be used as well as uncontrolled freezingimmediately at −20° C. or in liquid nitrogen.

In certain embodiments, cryopreserved cells are thawed and washed asdescribed herein and allowed to rest for one hour at room temperatureprior to activation using the methods of the present invention.

Also contemplated in the context of the invention is the collection ofblood samples or apheresis product from a subject at a time period priorto when the expanded cells as described herein might be needed. As such,the source of the cells to be expanded can be collected at any timepoint necessary, and desired cells, such as T cells, isolated and frozenfor later use in T cell therapy for any number of diseases or conditionsthat would benefit from T cell therapy, such as those described herein.In one embodiment a blood sample or an apheresis is taken from agenerally healthy subject. In certain embodiments, a blood sample or anapheresis is taken from a generally healthy subject who is at risk ofdeveloping a disease, but who has not yet developed a disease, and thecells of interest are isolated and frozen for later use. In certainembodiments, the T cells may be expanded, frozen, and used at a latertime. In certain embodiments, samples are collected from a patientshortly after diagnosis of a particular disease as described herein butprior to any treatments. In a further embodiment, the cells are isolatedfrom a blood sample or an apheresis from a subject prior to any numberof relevant treatment modalities, including but not limited to treatmentwith agents such as natalizumab, efalizumab, antiviral agents,chemotherapy, radiation, immunosuppressive agents, such as cyclosporin,azathioprine, methotrexate, mycophenolate, and FK506, antibodies, orother immunoablative agents such as CAMPATH, anti-CD3 antibodies,cytoxan, fludarabine, cyclosporin, FK506, rapamycin, mycophenolic acid,steroids, FR901228, and irradiation. These drugs inhibit either thecalcium dependent phosphatase calcineurin (cyclosporine and FK506) orinhibit the p70S6 kinase that is important for growth factor inducedsignaling (rapamycin). (Liu et al., Cell 66:807-815, 1991; Henderson etal., Immun. 73:316-321, 1991; Bierer et al., Curr. Opin. Immun.5:763-773, 1993). In a further embodiment, the cells are isolated for apatient and frozen for later use in conjunction with (e.g., before,simultaneously or following) bone marrow or stem cell transplantation, Tcell ablative therapy using either chemotherapy agents such as,fludarabine, external-beam radiation therapy (XRT), cyclophosphamide, orantibodies such as OKT3 or CAMPATH. In another embodiment, the cells areisolated prior to and can be frozen for later use for treatmentfollowing B-cell ablative therapy, e.g., Rituxan.

In a further embodiment of the present invention, T cells are obtainedfrom a patient directly following treatment. In this regard, it has beenobserved that following certain cancer treatments, in particulartreatments with drugs that damage the immune system, shortly aftertreatment during the period when patients would normally be recoveringfrom the treatment, the quality of T cells obtained may be optimal orimproved for their ability to expand ex vivo. Likewise, following exvivo manipulation using the methods described herein, these cells may bein a preferred state for enhanced engraftment and in vivo expansion.Thus, it is contemplated within the context of the present invention tocollect blood cells, including T cells, dendritic cells, or other cellsof the hematopoietic lineage, during this recovery phase. Further, incertain embodiments, mobilization (for example, mobilization withGM-CSF) and conditioning regimens can be used to create a condition in asubject wherein repopulation, recirculation, regeneration, and/orexpansion of particular cell types is favored, especially during adefined window of time following therapy. Illustrative cell typesinclude T cells, B cells, dendritic cells, and other cells of the immunesystem.

Activation and Expansion of T Cells

T cells are activated and expanded generally using methods as described,for example, in U.S. Pat. Nos. 6,352,694; 6,534,055; 6,905,680;6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318;7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514;6,867,041; and U.S. Patent Application Publication No. 20060121005.

Generally, the T cells of the invention are expanded by contact with asurface having attached thereto an agent that stimulates a CD3/TCRcomplex associated signal and a ligand that stimulates a co-stimulatorymolecule on the surface of the T cells. In particular, T cellpopulations may be stimulated as described herein, such as by contactwith an anti-CD3 antibody, or antigen-binding fragment thereof, or ananti-CD2 antibody immobilized on a surface, or by contact with a proteinkinase C activator (e.g., bryostatin) in conjunction with a calciumionophore. For co-stimulation of an accessory molecule on the surface ofthe T cells, a ligand that binds the accessory molecule is used. Forexample, a population of T cells can be contacted with an anti-CD3antibody and an anti-CD28 antibody, under conditions appropriate forstimulating proliferation of the T cells. To stimulate proliferation ofeither CD4⁺ T cells or CD8⁺ T cells, an anti-CD3 antibody and ananti-CD28 antibody. Examples of an anti-CD28 antibody include 9.3, B-T3,XR-CD28 (Diaclone, Besancon, France) can be used as can other methodscommonly known in the art (Berg et al., Transplant Proc.30(8):3975-3977, 1998; Haanen et al., J. Exp. Med. 190(9):13191328,1999; Garland et al., J. Immunol Meth. 227(1-2):53-63, 1999).

In certain embodiments, the primary stimulatory signal and theco-stimulatory signal for the T cell may be provided by differentprotocols. For example, the agents providing each signal may be insolution or coupled to a surface. When coupled to a surface, the agentsmay be coupled to the same surface (i.e., in “cis” formation) or toseparate surfaces (i.e., in “trans” formation). Alternatively, one agentmay be coupled to a surface and the other agent in solution. In oneembodiment, the agent providing the co-stimulatory signal is bound to acell surface and the agent providing the primary activation signal is insolution or coupled to a surface. In certain embodiments, both agentscan be in solution. In another embodiment, the agents may be in solubleform, and then cross-linked to a surface, such as a cell expressing Fcreceptors or an antibody or other binding agent which will bind to theagents. In this regard, see for example, U.S. Patent ApplicationPublication Nos. 20040101519 and 20060034810 for artificial antigenpresenting cells (aAPCs) that are contemplated for use in activating andexpanding T cells in the present invention.

In one embodiment, the two agents are immobilized on beads, either onthe same bead, i.e., “cis,” or to separate beads, i.e., “trans.” By wayof example, the agent providing the primary activation signal is ananti-CD3 antibody or an antigen-binding fragment thereof and the agentproviding the co-stimulatory signal is an anti-CD28 antibody orantigen-binding fragment thereof; and both agents are co-immobilized tothe same bead in equivalent molecular amounts. In one embodiment, a 1:1ratio of each antibody bound to the beads for CD4⁺ T cell expansion andT cell growth is used. In certain aspects of the present invention, aratio of anti CD3:CD28 antibodies bound to the beads is used such thatan increase in T cell expansion is observed as compared to the expansionobserved using a ratio of 1:1. In one particular embodiment an increaseof from about 1 to about 3 fold is observed as compared to the expansionobserved using a ratio of 1:1. In one embodiment, the ratio of CD3:CD28antibody bound to the beads ranges from 100:1 to 1:100 and all integervalues there between. In one aspect of the present invention, moreanti-CD28 antibody is bound to the particles than anti-CD3 antibody,i.e., the ratio of CD3:CD28 is less than one. In certain embodiments ofthe invention, the ratio of anti CD28 antibody to anti CD3 antibodybound to the beads is greater than 2:1. In one particular embodiment, a1:100 CD3:CD28 ratio of antibody bound to beads is used. In anotherembodiment, a 1:75 CD3:CD28 ratio of antibody bound to beads is used. Ina further embodiment, a 1:50 CD3:CD28 ratio of antibody bound to beadsis used. In another embodiment, a 1:30 CD3:CD28 ratio of antibody boundto beads is used. In one preferred embodiment, a 1:10 CD3:CD28 ratio ofantibody bound to beads is used. In another embodiment, a 1:3 CD3:CD28ratio of antibody bound to the beads is used. In yet another embodiment,a 3:1 CD3:CD28 ratio of antibody bound to the beads is used.

Ratios of particles to cells from 1:500 to 500:1 and any integer valuesin between may be used to stimulate T cells or other target cells. Asthose of ordinary skill in the art can readily appreciate, the ratio ofparticles to cells may depend on particle size relative to the targetcell. For example, small sized beads could only bind a few cells, whilelarger beads could bind many. In certain embodiments the ratio of cellsto particles ranges from 1:100 to 100:1 and any integer valuesin-between and in further embodiments the ratio comprises 1:9 to 9:1 andany integer values in between, can also be used to stimulate T cells.The ratio of anti-CD3- and anti-CD28-coupled particles to T cells thatresult in T cell stimulation can vary as noted above, however certainpreferred values include 1:100, 1:50, 1:40, 1:30, 1:20, 1:10, 1:9, 1:8,1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1,9:1, 10:1, and 15:1 with one preferred ratio being at least 1:1particles per T cell. In one embodiment, a ratio of particles to cellsof 1:1 or less is used. In one particular embodiment, a preferredparticle: cell ratio is 1:5. In further embodiments, the ratio ofparticles to cells can be varied depending on the day of stimulation.For example, in one embodiment, the ratio of particles to cells is from1:1 to 10:1 on the first day and additional particles are added to thecells every day or every other day thereafter for up to 10 days, atfinal ratios of from 1:1 to 1:10 (based on cell counts on the day ofaddition). In one particular embodiment, the ratio of particles to cellsis 1:1 on the first day of stimulation and adjusted to 1:5 on the thirdand fifth days of stimulation. In another embodiment, particles areadded on a daily or every other day basis to a final ratio of 1:1 on thefirst day, and 1:5 on the third and fifth days of stimulation. Inanother embodiment, the ratio of particles to cells is 2:1 on the firstday of stimulation and adjusted to 1:10 on the third and fifth days ofstimulation. In another embodiment, particles are added on a daily orevery other day basis to a final ratio of 1:1 on the first day, and 1:10on the third and fifth days of stimulation. One of skill in the art willappreciate that a variety of other ratios may be suitable for use in thepresent invention. In particular, ratios will vary depending on particlesize and on cell size and type.

In further embodiments of the present invention, the cells, such as Tcells, are combined with agent-coated beads, the beads and the cells aresubsequently separated, and then the cells are cultured. In analternative embodiment, prior to culture, the agent-coated beads andcells are not separated but are cultured together. In a furtherembodiment, the beads and cells are first concentrated by application ofa force, such as a magnetic force, resulting in increased ligation ofcell surface markers, thereby inducing cell stimulation.

By way of example, cell surface proteins may be ligated by allowingparamagnetic beads to which anti-CD3 and anti-CD28 are attached (3×28beads) to contact the T cells. In one embodiment the cells (for example,10⁴ to 10⁹ T cells) and beads (for example, DYNABEADS® M-450 CD3/CD28 Tparamagnetic beads at a ratio of 1:1) are combined in a buffer, forexample PBS (without divalent cations such as, calcium and magnesium).Again, those of ordinary skill in the art can readily appreciate anycell concentration may be used. For example, the target cell may be veryrare in the sample and comprise only 0.01% of the sample or the entiresample (i.e., 100%) may comprise the target cell of interest.Accordingly, any cell number is within the context of the presentinvention. In certain embodiments, it may be desirable to significantlydecrease the volume in which particles and cells are mixed together(i.e., increase the concentration of cells), to ensure maximum contactof cells and particles. For example, in one embodiment, a concentrationof about 2 billion cells/ml is used. In another embodiment, greater than100 million cells/ml is used. In a further embodiment, a concentrationof cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells/ml isused. In yet another embodiment, a concentration of cells from 75, 80,85, 90, 95, or 100 million cells/ml is used. In further embodiments,concentrations of 125 or 150 million cells/ml can be used. Using highconcentrations can result in increased cell yield, cell activation, andcell expansion. Further, use of high cell concentrations allows moreefficient capture of cells that may weakly express target antigens ofinterest, such as CD28-negative T cells. Such populations of cells mayhave therapeutic value and would be desirable to obtain in certainembodiments. For example, using high concentration of cells allows moreefficient selection of CD8+ T cells that normally have weaker CD28expression.

In one embodiment of the present invention, the mixture may be culturedfor several hours (about 3 hours) to about 14 days or any hourly integervalue in between. In another embodiment, the mixture may be cultured for21 days. In one embodiment of the invention the beads and the T cellsare cultured together for about eight days. In another embodiment, thebeads and T cells are cultured together for 2-3 days. Several cycles ofstimulation may also be desired such that culture time of T cells can be60 days or more. Conditions appropriate for T cell culture include anappropriate media (e.g., Minimal Essential Media or RPMI Media 1640 or,X-vivo 15, (Lonza)) that may contain factors necessary for proliferationand viability, including serum (e.g., fetal bovine or human serum),interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12,IL-15, TGFβ, and TNF-α or any other additives for the growth of cellsknown to the skilled artisan. Other additives for the growth of cellsinclude, but are not limited to, surfactant, plasmanate, and reducingagents such as N-acetyl-cysteine and 2-mercaptoethanol. Media caninclude RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15, and X-Vivo20, Optimizer, with added amino acids, sodium pyruvate, and vitamins,either serum-free or supplemented with an appropriate amount of serum(or plasma) or a defined set of hormones, and/or an amount ofcytokine(s) sufficient for the growth and expansion of T cells.Antibiotics, e.g., penicillin and streptomycin, are included only inexperimental cultures, not in cultures of cells that are to be infusedinto a subject. The target cells are maintained under conditionsnecessary to support growth, for example, an appropriate temperature(e.g., 37° C.) and atmosphere (e.g., air plus 5% CO₂).

T cells that have been exposed to varied stimulation times may exhibitdifferent characteristics. For example, typical blood or apheresedperipheral blood mononuclear cell products have a helper T cellpopulation (T_(H), CD4⁺) that is greater than the cytotoxic orsuppressor T cell population (T_(C), CD8⁺). Ex vivo expansion of T cellsby stimulating CD3 and CD28 receptors produces a population of T cellsthat prior to about days 8-9 consists predominately of T_(H) cells,while after about days 8-9, the population of T cells comprises anincreasingly greater population of T_(C) cells. Accordingly, dependingon the purpose of treatment, infusing a subject with a T cell populationcomprising predominately of T_(H) cells may be advantageous. Similarly,if an antigen-specific subset of T_(C) cells has been isolated it may bebeneficial to expand this subset to a greater degree.

Further, in addition to CD4 and CD8 markers, other phenotypic markersvary significantly, but in large part, reproducibly during the course ofthe cell expansion process. Thus, such reproducibility enables theability to tailor an activated T cell product for specific purposes.

Therapeutic Application

In one aspect, the invention includes a method for treating anautoimmune disease in a subject. The method comprises: administering tothe subject an effective amount of a genetically modified T cellcomprising an isolated nucleic acid sequence encoding a chimericautoantibody receptor (CAAR), wherein the isolated nucleic acid sequencecomprises a nucleic acid sequence of an extracellular domain comprisingan autoantigen or fragment thereof, a nucleic acid sequence of atransmembrane domain, and a nucleic acid sequence of an intracellularsignaling domain, thereby treating the autoimmune disease in thesubject.

In one embodiment, the autoimmune disease is selected from pemphigusvulgaris paraneoplastic pemphigus, or pemphigus foliaceus. In anotherembodiment, the subject is a human.

Without wishing to be bound by any particular theory, theanti-autoantibody immune response elicited by the CAAR-modified T cellsmay be an active or a passive immune response. In yet anotherembodiment, the modified T cell targets a B cell. For example,autoantibody expressing B cells may be susceptible to indirectdestruction by CAAR-redirected T cells that have previously reactedagainst adjacent autoantibody-expressing cells.

In one embodiment, the fully-human CAAR-genetically modified T cells ofthe invention may be a type of vaccine for ex vivo immunization and/orin vivo therapy in a mammal. In one embodiment, the mammal is a human.

With respect to ex vivo immunization, at least one of the followingoccurs in vitro prior to administering the cell into a mammal: i)expansion of the cells, ii) introducing a nucleic acid encoding a CAARto the cells or iii) cryopreservation of the cells.

Ex vivo procedures are well known in the art and are discussed morefully below. Briefly, cells are isolated from a mammal (e.g., a human)and genetically modified (i.e., transduced or transfected in vitro) witha vector expressing a CAAR disclosed herein. The CAAR-modified cell canbe administered to a mammalian recipient to provide a therapeuticbenefit. The mammalian recipient may be a human and the CAAR-modifiedcell can be autologous with respect to the recipient. Alternatively, thecells can be allogeneic, syngeneic or xenogeneic with respect to therecipient.

The procedure for ex vivo expansion of hematopoietic stem and progenitorcells is described in U.S. Pat. No. 5,199,942, incorporated herein byreference, can be applied to the cells of the present invention. Othersuitable methods are known in the art, therefore the present inventionis not limited to any particular method of ex vivo expansion of thecells. Briefly, ex vivo culture and expansion of T cells comprises: (1)collecting CD34+ hematopoietic stem and progenitor cells from a mammalfrom peripheral blood harvest or bone marrow explants; and (2) expandingsuch cells ex vivo. In addition to the cellular growth factors describedin U.S. Pat. No. 5,199,942, other factors such as flt3-L, IL-1, IL-3 andc-kit ligand, can be used for culturing and expansion of the cells.

In addition to using a cell-based vaccine in terms of ex vivoimmunization, the present invention also includes compositions andmethods for in vivo immunization to elicit an immune response directedagainst an antigen in a patient.

Generally, the cells activated and expanded as described herein may beutilized in the treatment and prevention of diseases that arise inindividuals who are immunocompromised. In particular, the CAAR-modifiedT cells of the invention are used in the treatment of diseases,disorders and conditions associated with expression of autoantibodies.In certain embodiments, the cells of the invention are used in thetreatment of patients at risk for developing autoimmune diseases,disorders and conditions associated with expression of autoantibodies.Thus, the present invention provides methods for the treatment orprevention of autoimmune diseases, disorders and conditions associatedwith expression of autoantibodies comprising administering to a subjectin need thereof, a therapeutically effective amount of the CAAR-modifiedT cells of the invention.

The CAAR-modified T cells of the present invention may be administeredeither alone, or as a pharmaceutical composition in combination withdiluents and/or with other components such as IL-2 or other cytokines orcell populations. Briefly, pharmaceutical compositions of the presentinvention may comprise a target cell population as described herein, incombination with one or more pharmaceutically or physiologicallyacceptable carriers, diluents or excipients. Such compositions maycomprise buffers such as neutral buffered saline, phosphate bufferedsaline and the like; carbohydrates such as glucose, mannose, sucrose ordextrans, mannitol; proteins; polypeptides or amino acids such asglycine; antioxidants; chelating agents such as EDTA or glutathione;adjuvants (e.g., aluminum hydroxide); and preservatives. Compositions ofthe present invention are in one aspect formulated for intravenousadministration.

Pharmaceutical compositions of the present invention may be administeredin a manner appropriate to the disease to be treated (or prevented). Thequantity and frequency of administration will be determined by suchfactors as the condition of the patient, and the type and severity ofthe patient's disease, although appropriate dosages may be determined byclinical trials.

When “an immunologically effective amount,” “an anti-autoantibodyeffective amount,” “an autoimmune disease-inhibiting effective amount,”or “therapeutic amount” is indicated, the precise amount of thecompositions of the present invention to be administered can bedetermined by a physician with consideration of individual differencesin age, weight, tumor size, extent of infection or metastasis, andcondition of the patient (subject). It can generally be stated that apharmaceutical composition comprising the T cells described herein maybe administered at a dosage of 10⁴ to 10⁹ cells/kg body weight, in someinstances 10⁵ to 10⁶ cells/kg body weight, including all integer valueswithin those ranges. T cell compositions may also be administeredmultiple times at these dosages. The cells can be administered by usinginfusion techniques that are commonly known in immunotherapy (see, e.g.,Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimaldosage and treatment regime for a particular patient can readily bedetermined by one skilled in the art of medicine by monitoring thepatient for signs of disease and adjusting the treatment accordingly.

In certain embodiments, activated T cells are administered to a subject.Subsequent to administration, blood is redrawn or apheresis isperformed, and T cells are activated and expanded therefrom using themethods described here, and are then reinfused back into the patient.This process can be carried out multiple times every few weeks. Incertain embodiments, T cells can be activated from blood draws of from10 cc to 400 cc. In certain embodiments, T cells are activated fromblood draws of 20 cc, 30 cc, 40 cc, 50 cc, 60 cc, 70 cc, 80 cc, 90 cc,or 100 cc. Not to be bound by theory, using this multiple blooddraw/multiple reinfusion protocol, may select out certain populations ofT cells.

Administration of the cells of the invention may be carried out usingany convenient means, including by aerosol inhalation, injection,ingestion, transfusion, implantation or transplantation. Thecompositions described herein may be administered to a patienttransarterially, subcutaneously, intradermally, intratumorally,intranodally, intramedullary, intramuscularly, by intravenous (i. v.)injection, or intraperitoneally. In one embodiment, the T cellcompositions of the present invention are administered to a patient byintradermal or subcutaneous injection. In another embodiment, the T cellcompositions of the present invention are administered by i.v.injection. The compositions of T cells may be injected directly into atumor, lymph node, or site of infection.

In certain embodiments of the present invention, cells activated andexpanded using the methods described herein, or other methods known inthe art where T cells are expanded to therapeutic levels, areadministered to a patient in conjunction with (e.g., before,simultaneously or following) any number of relevant treatmentmodalities, including but not limited to treatment with agents such asantiviral therapy, cidofovir and interleukin-2, Cytarabine (also knownas ARA-C) or natalizumab treatment for MS patients or efalizumabtreatment for psoriasis patients or other treatments for PML patients.In further embodiments, the T cells of the invention may be used incombination with chemotherapy, radiation, immunosuppressive agents, suchas cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506,antibodies, or other immunoablative agents such as CAM PATH, anti-CD3antibodies or other antibody therapies, cytoxin, fludarabine,cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228,cytokines, and irradiation. These drugs inhibit either the calciumdependent phosphatase calcineurin (cyclosporine and FK506) or inhibitthe p70S6 kinase that is important for growth factor induced signaling(rapamycin). (Liu et al., Cell 66:807-815, 1991; Henderson et al.,Immun. 73:316-321, 1991; Bierer et al., Curr. Opin. Immun. 5:763-773,1993). In a further embodiment, the cell compositions of the presentinvention are administered to a patient in conjunction with (e.g.,before, simultaneously or following) bone marrow transplantation, T cellablative therapy using either chemotherapy agents such as, fludarabine,external-beam radiation therapy (XRT), cyclophosphamide, or antibodiessuch as OKT3 or CAMPATH. In another embodiment, the cell compositions ofthe present invention are administered following B-cell ablative therapysuch as agents that react with CD20, e.g., Rituxan. For example, in oneembodiment, subjects may undergo standard treatment with high dosechemotherapy followed by peripheral blood stem cell transplantation. Incertain embodiments, following the transplant, subjects receive aninfusion of the expanded immune cells of the present invention. In anadditional embodiment, expanded cells are administered before orfollowing surgery.

The dosage of the above treatments to be administered to a patient willvary with the precise nature of the condition being treated and therecipient of the treatment. The scaling of dosages for humanadministration can be performed according to art-accepted practices. Thedose for CAMPATH, for example, will generally be in the range 1 to about100 mg for an adult patient, usually administered daily for a periodbetween 1 and 30 days. The preferred daily dose is 1 to 10 mg per dayalthough in some instances larger doses of up to 40 mg per day may beused (described in U.S. Pat. No. 6,120,766).

EXPERIMENTAL EXAMPLES

The invention is further described in detail by reference to thefollowing experimental examples. These examples are provided forpurposes of illustration only, and are not intended to be limitingunless otherwise specified. Thus, the invention should in no way beconstrued as being limited to the following examples, but rather, shouldbe construed to encompass any and all variations which become evident asa result of the teaching provided herein.

Without further description, it is believed that one of ordinary skillin the art can, using the preceding description and the followingillustrative examples, make and utilize the compounds of the presentinvention and practice the claimed methods. The following workingexamples therefore, specifically point out the preferred embodiments ofthe present invention, and are not to be construed as limiting in anyway the remainder of the disclosure.

The Materials and Methods used in the performance of the experimentsdisclosed herein are now described.

CAAR Constructs

The desmoglein 3 (Dsg3) CAAR was cloned by PCR amplification from humancDNA with specific primers

-   -   a) for the signal peptide of human CD8 alpha (fragment A)        (forward: 5′CTAGCAGGATCCGCCACCATGGCCTTACCAGTGACCG (SEQ ID NO:17)        (adding a Kozak sequence and a BamHI restriction site), reverse:        5′ TCTATTCGCAATTCCGGCCTGGCGGCG (SEQ ID NO:18), overlapping into        the propeptide of human Dsg3),    -   b) the signal peptide of the human CD8 hinge and transmembrane        region (fragment C) (forward:        5′CTCAGGGAGGAAGCCCACCACGACGCCAGCGCCGC (SEQ ID NO:19) (5′ overlap        from EC5 of human Dsg3), reverse:        5′CCCCGTTTGGTGATAACCAGTGACAGGAGAAGG (SEQ ID NO:20) (5′overlap        into the human CD137 signal transduction domain)),    -   c) the human CD137 signal transduction domain (fragment D)        (forward: 5′CTGGTTATCACCAAACGGGGCAGAAAGAAACTCC (SEQ ID NO:21),        reverse: 5′TTCACTCTCAGTTCACATCCTCCTTCTTCTTCTTCTGG (SEQ ID NO:22)        (overlapping into the human CD247 (aka CD3 zeta) signal        transduction domain)),    -   d) and the human CD3 zeta signal transduction domain        (fragment E) (forward: 5′GATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGC (SEQ        ID NO:23), reverse: 5′ GGTTGATTGTCGACGCGGATCTTAGCGAGGGGGC (SEQ        ID NO:24) (adding a SalI site after the TAA stop codon)),    -   e) for expression in a non-lentiviral vector plasmid the BamHI        site was replaced with a XhoI site and the SalI site was        replaced with a BamHI site.

The exon-encoding sequence of human Dsg3 (fragment B) was amplified fromthe plasmid DN653 (a gift from Prof. Amagai, Keio University, Tokyo,Japan) with the primers forward 5′CCAGGCCGGAATTGCGAATAGAGACTAAAGG (SEQID NO:25) and reverse 5′CGTGGTGGGCTTCCTCCCTGAGTGCGGCC (SEQ ID NO:26), sothat an overlap with the 5′ located sequence of the signal peptide ofCD8 and the 3′ located CD8 hinge became possible.

After verification of the correct size of the PCR products, thefragments were purified (Promega wizard SV) and subjected to extensionoverlap PCRs, joining fragment A with B as well as C with D. The joinedfragment CD was extended with another overlap-extension PCR withfragment E and finally the fragments AB and CDE were PCR-conjugated. AllPCR reactions were performed with Q5 hot start polymerase (New EnglandBiolabs) according to the manufacturer's recommendation. The final 2.5kB long PCR product was subjected to a gel purification and digestedeither with BamHI-SalI (cloning into lentiviral vector plasmid) orXhoI-BamHI (cloning into non-lentiviral expression plasmid, namelypCEP4, life technologies).

To facilitate constitutive expression under a strong human promoter thatis not prone to silencing in lymphoid cells, the Dsg3 CAAR was clonedinto a 3rd generation HIV1-basedlentiviral vector plasmid, namelypRRLSIN.cPPT.PGK-GFP.WPRE (addgene 12252). Since previous studies hadshown a favorable expression under the EF1alpha compared to the PGKpromoter, we PCR amplified the EF1alpha promoter from human genomic DNAusing the primers forward 5′GGATCCTGCTAGACTCACGACACCTGAAATGGAAG (SEQ IDNO:27) and reverse 5′ GAGGAGGTCGACATTCGTGAGGCTCCGGTGCCCGTC (SEQ IDNO:28). The PGK promoter was replaced with the EF1alpha promoter bydigesting the PCR product with SalI and BamHI and the plasmid with XhoIand BamHI. The compatible ends of SalI and XhoI result in a deletion ofthe XhoI and SalI sites, so that the plasmid retains the unique BamHIand SalI sites flanking the GFP that was replaced with the Dsg3 CAAR bydigestion and ligation.

Shortened versions of the Dsg3 CAAR were cloned into the same plasmidbackbone using BamHI and SalI. To facilitate high surface expression theshortened versions were codon optimized using a codon adaptationindex-based algorithm (geneart, life technologies) and synthesized asdouble-stranded DNA fragments (geneart, life technologies). In theseconstructs the CD8 hinge region was replaced with an 13 amino acid longflexible GS-linker, providing a unique NheI site that could be used toinsert different Dsg3 encoding fragments between the Kozak sequence(with BamHI) and the GS-linker (with NheI). The complete extracellularDsg3 was cloned into this cloning site and from the derived plasmidvarious versions of Dsg3 were produced with the following primers:

BamHI-CD8 signal peptide - Dsg3EC1-5-NheI: BamHI.CD8.for:(SEQ ID NO: 29) GAGGAGGAGGGATCCGCCACC EC5.NheI.rev: (SEQ ID NO: 30)CCTCCGCCGCCGCTAGCTCTGCC BamHI-CD8 signal peptide - Dsg3EC1-4-NheIBamHI.CD8.for: (SEQ ID NO: 29) GAGGAGGAGGGATCCGCCACC EC4.NheI.rev:(SEQ ID NO: 31) CCTCCGCCGCCGCTAGCCTTTTCCAGCACGGCGGBamHI-CD8 signal peptide - Dsg3EC1-3-NheI: BamHI.CD8.for:(SEQ ID NO: 29) GAGGAGGAGGGATCCGCCACC EC3.NheI.rev: (SEQ ID NO: 32)TCTCCTCGCTAGCGAAGGCAATGCCC BamHI-CD8 signal peptide - Dsg3EC1-2-NheI:BamHI.CD8.for: (SEQ ID NO: 29) GAGGAGGAGGGATCCGCCACC EC2.NheI.rev:(SEQ ID NO: 33) TCCGCCGCCGCTAGCCCGGAACATAGGGAAGTTGTCG

In order to clone a CAAR that presents the EC2-3 of Dsg3, theEC3.NheI.rev primer was used in combination with a primer for the 5′sequence of the EC2 (5′AAGCGGCGGCAGAAACGCATCCTGGACATCAACGACAACC) (SEQ IDNO:34); the resulting EC2-3 sequence was PCR-conjugated with the CD8signal peptide (previously amplified with the overlapping reverse primer5′ GATGCGTTTCTGCCGCCGCTTGGCCTGCTGCATTGTC (SEQ ID NO:35) and theBamHI.CD8 forward primer (see above)). The sequence of the completeEC1-5 CAAR construct is as follows:

(SEQ ID NO: 36) ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCCCTGCTGCTGCATGTGCCAGACCTGGCTCCGAGCTGCGGATCGAGACAAAGGGCCAGTACGACGAGGAAGAGATGACAATGCAGCAGGCCAAGCGGCGGCAGAAACGCGAGTGGGTCAAGTTCGCCAAGCCCTGCAGAGAGGGCGAGGACAACAGCAAGCGGAACCCTATCGCCAAGATCACCAGCGACTACCAGGCCACCCAGAAGATCACCTACCGGATCAGCGGCGTGGGCATCGACCAGCCCCCTTTCGGCATCTTCGTGGTGGACAAGAACACCGGCGACATCAACATCACCGCCATCGTGGACAGAGAGGAAACCCCCAGCTTCCTGATCACCTGTCGGGCCCTGAATGCCCAGGGCCTGGACGTGGAAAAGCCCCTGATCCTGACCGTGAAGATCCTGGACATCAACGACAACCCCCCCGTGTTCAGCCAGCAGATCTTCATGGGCGAGATCGAGGAAAACAGCGCCAGCAACAGCCTCGTGATGATCCTGAACGCCACCGACGCCGACGAGCCCAACCACCTGAATAGCAAGATCGCCTTCAAGATCGTGTCCCAGGAACCCGCCGGAACCCCCATGTTCCTGCTGAGCAGAAATACCGGCGAAGTGCGGACCCTGACCAACAGCCTGGATAGAGAGCAGGCCAGCAGCTACCGGCTGGTGGTGTCTGGCGCTGACAAGGATGGCGAGGGCCTGAGCACACAGTGCGAGTGCAACATCAAAGTGAAGGACGTGAACGACAACTTCCCTATGTTCCGGGACAGCCAGTACAGCGCCCGGATCGAAGAGAACATCCTGAGCAGCGAGCTGCTGCGGTTCCAAGTGACCGACCTGGACGAAGAGTACACCGACAACTGGCTAGCCGTGTACTTCTTCACCAGCGGCAACGAGGGCAATTGGTTCGAGATCCAGACCGACCCCCGGACCAATGAGGGCATCCTGAAGGTCGTGAAGGCCCTGGACTACGAGCAGCTGCAGAGCGTGAAGCTGTCTATCGCCGTGAAGAACAAGGCCGAGTTCCACCAGTCCGTGATCAGCCGGTACAGAGTGCAGAGCACCCCCGTGACCATCCAAGTGATCAACGTGCGCGAGGGCATTGCCTTCAGACCCGCCAGCAAGACCTTCACCGTGCAGAAGGGCATCAGCAGCAAGAAACTGGTGGACTACATCCTGGGCACCTATCAGGCCATCGACGAGGACACCAACAAAGCCGCCTCCAACGTGAAATACGTGATGGGCCGGAACGACGGCGGCTACCTGATGATCGATTCCAAGACCGCCGAGATCAAGTTCGTGAAGAATATGAACCGGGACTCCACCTTCATCGTGAACAAGACCATCACAGCCGAGGTGCTGGCCATCGATGAGTATACCGGCAAGACCAGCACCGGCACCGTGTACGTGCGGGTGCCCGACTTCAACGATAACTGCCCTACCGCCGTGCTGGAAAAGGACGCCGTGTGTAGCAGCAGCCCCAGCGTGGTGGTGTCCGCCAGAACCCTGAACAACCGGTACACCGGCCCCTACACCTTCGCCCTGGAAGATCAGCC TGTGAAGCTGCCCGCCGTGTGGTCCATCACCACACTGAATGCCACCAGCGCCCTGCTGAGAGCCCAGGAACAGATTCCCCCTGGCGTGTACCACATCAGCCTGGTGCTGACCGACAGCCAGAACAACAGATGCGAGATGCCCCGGTCCCTGACCCTGGAAGTGTGCCAGTGCGACAACAGAGGCATCTGCGGCACCAGCTACCCTACCACCTCTCCCGGCACCAGATACGGCAGACCTCACAGCGGCAGAGCTAGCGGCGGCGGAGGAAGCGGAGGCGGAGGATCTAGCGGCATCTACATCTGGGCCCCTCTGGCCGGAACATGCGGAGTGCTGCTGCTGAGCCTCGTGATCACCCTGTACTGCAAGAGAGGCCGGAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCATGCGGCCCGTGCAGACCACCCAGGAAGAGGACGGCTGCAGCTGTCGGTTCCCCGAGGAAGAAGAAGGCGGCTGCGAACTGAGAGTGAAGTTCAGCAGAAGCGCCGACGCCCCTGCCTACCAGCAGGGACAGAACCAGCTGTACAACGAGCTGAACCTGGGCAGACGGGAAGAGTACGACGTGCTGGACAAGCGGAGAGGCAGGGACCCTGAGATGGGCGGCAAGCCCAGAAGAAAGAACCCCCAGGAAGGCCTGTATAACGAACTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGAATGAAGGGCGAGCGGAGAAGAGGCAAGGGCCACGACGGACTGTACCAGGGACTGAGCACCGCCACCAAGGACACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCTAGATAA.

The desmoglein 1 (Dsg1) CAAR was cloned by PCR amplification from humancDNA using specific primers

The sequence of the complete CAAR construct is as follows:

Dsg1 CAAR EC1-3 Nucleotide Sequence (Extracellular Portion up toGS-Linker, Transmembrane and Cytoplasmic Domains Same as for Dsg3 CAAR)

(SEQ ID NO: 37) ATGGCACTTCCAGTGACCGCTCTGCTCCTGCCACTGGCCCTGCTGCTCCACGCTGCCCGCCCGGGCAGCGAGTTCAGGATCCAAGTCAGGGATTATAATACTAAAAACGGTACCATCAAGTGGCATTCCATACGCAGGCAGAAAAGGGAGTGGATTAAGTTTGCTGCCGCGTGCCGGGAGGGTGAAGACAATAGCAAACGGAATCCCATTGCAAAGATACATAGCGATTGCGCTGCCAATCAGCAGGTTACATATCGAATCTCCGGCGTGGGGATTGACCAGCCTCCTTATGGCATTTTCGTCATTAACCAAAAGACTGGCGAGATAAATATCACATCAATTGTGGACCGGGAAGTGACGCCGTTTTTTATCATCTACTGTAGAGCTCTGAACTCCATGGGCCAGGATCTGGAAAGGCCACTGGAGCTGAGGGTCAGGGTCCTTGACATCAATGACAATCCCCCCGTCTTTTCCATGGCCACGTTCGCCGGACAGATTGAGGAAAATAGCAATGCCAATACACTGGTGATGATCCTGAACGCTACCGACGCTGACGAGCCGAATAATCTGAACAGTAAAATTGCTTTTAAGATCATTCGGCAGGAGCCATCAGACAGCCCAATGTTTATCATTAACAGAAACACCGGAGAGATCCGCACAATGAACAATTTCCTGGATAGGGAACAGTATGGACAGTATGCACTCGCTGTTCGGGGCTCCGACCGGGACGGTGGAGCTGATGGCATGAGTGCCGAGTGCGAGTGCAATATCAAGATACTCGACGTAAATGATAATATTCCATACATGGAACAGAGCTCTTACACTATCGAGATCCAGGAGAATACTCTCAACTCTAATCTTCTTGAAATTAGAGTGATTGATCTCGACGAGGAATTTTCTGCCAATTGGATGGCTGTCATCTTCTTTATTAGTGGTAACGAGGGTAACTGGTTCGAGATAGAAATGAATGAAAGGACAAATGTGGGAATCTTGAAGGTGGTTAAACCACTGGACTACGAAGCAATGCAATCACTCCAGCTGTCAATAGGCGTCAGAAATAAGGCGGAGTTCCATCACTCCATTATGTCCCAGTATAAATTGAAAGCCAGTGCCATAAGCGTAACCGTGTTGAACGTGATAGAAGGGCCTG TTTTTGCATCCGGADsg1 CAAR EC1-3 Amino Acid Sequence (Extracellular Portion up toGS-Linker, Transmembrane and Cytoplasmic Domains Same as for Dsg3 CAAR)

(SEQ ID NO: 38) MALPVTALLLPLALLLHAARPGSEFRIQVRDYNTKNGTIKWHSIRRQKREWIKFAAACREGEDNSKRNPIAKIHSDCAANQQVTYRISGVGIDQPPYGIFVINQKTGEINITSIVDREVTPFFIIYCRALNSMGQDLERPLELRVRVLDINDNPPVFSMATFAGQIEENSNANTLVMILNATDADEPNNLNSKIAFKIIRQEPSDSPMFIINRNTGEIRTMNNFLDREQYGQYALAVRGSDRDGGADGMSAECECNIKILDVNDNIPYMEQSSYTIEIQENTLNSNLLEIRVIDLDEEFSANWMAVIFFISGNEGNWFEIEMNERTNVGILKVVKPLDYEAMQSLQLSIGVRNKAEFEIFHHSIMSQYKLKASAISVTVLNVIEGPVFASG Dsg1 CAAR EC1-4 Nucleotide Sequence (Extracellular Portion up toGS-Linker, Transmembrane and Cytoplasmic Domains Same as for Dsg3 CAAR)

(SEQ ID NO: 39) ATGGCACTTCCAGTGACCGCTCTGCTCCTGCCACTGGCCCTGCTGCTCCACGCTGCCCGCCCGGGCAGCGAGTTCAGGATCCAAGTCAGGGATTATAATACTAAAAACGGTACCATCAAGTGGCATTCCATACGCAGGCAGAAAAGGGAGTGGATTAAGTTTGCTGCCGCGTGCCGGGAGGGTGAAGACAATAGCAAACGGAATCCCATTGCAAAGATACATAGCGATTGCGCTGCCAATCAGCAGGTTACATATCGAATCTCCGGCGTGGGGATTGACCAGCCTCCTTATGGCATTTTCGTCATTAACCAAAAGACTGGCGAGATAAATATCACATCAATTGTGGACCGGGAAGTGACGCCGTTTTTTATCATCTACTGTAGAGCTCTGAACTCCATGGGCCAGGATCTGGAAAGGCCACTGGAGCTGAGGGTCAGGGTCCTTGACATCAATGACAATCCCCCCGTCTTTTCCATGGCCACGTTCGCCGGACAGATTGAGGAAAATAGCAATGCCAATACACTGGTGATGATCCTGAACGCTACCGACGCTGACGAGCCGAATAATCTGAACAGTAAAATTGCTTTTAAGATCATTCGGCAGGAGCCATCAGACAGCCCAATGTTTATCATTAACAGAAACACCGGAGAGATCCGCACAATGAACAATTTCCTGGATAGGGAACAGTATGGACAGTATGCACTCGCTGTTCGGGGCTCCGACCGGGACGGTGGAGCTGATGGCATGAGTGCCGAGTGCGAGTGCAATATCAAGATACTCGACGTAAATGATAATATTCCATACATGGAACAGAGCTCTTACACTATCGAGATCCAGGAGAATACTCTCAACTCTAATCTTCTTGAAATTAGAGTGATTGATCTCGACGAGGAATTTTCTGCCAATTGGATGGCTGTCATCTTCTTTATTAGTGGTAACGAGGGTAACTGGTTCGAGATAGAAATGAATGAAAGGACAAATGTGGGAATCTTGAAGGTGGTTAAACCACTGGACTACGAAGCAATGCAATCACTCCAGCTGTCAATAGGCGTCAGAAATAAGGCGGAGTTCCATCACTCCATTATGTCCCAGTATAAATTGAAAGCCAGTGCCATAAGCGTAACCGTGTTGAACGTGATAGAAGGGCCTGTTTTTCGCCCTGGGTCCAAAACCTACGTTGTGACAGGAAACATGGGATCCAACGACAAAGTCGGCGACTTCGTCGCAACAGACCTGGACACCGGTCGCCCTTCCACAACTGTGCGGTACGTGATGGGAAACAATCCAGCCGACTTGTTGGCAGTCGATAGCAGGACAGGGAAGCTGACCCTTAAAAACAAGGTTACAAAAGAACAATATAACATGCTGGGCGGCAAATATCAGGGAACCATTTTGTCAATCGACGACAACCTGCAGCGCACGTGCACGGGGACGATCAACATCAACATCCAGAGCTTTGGGAATGACGATAGAACCAACACAGAGCCCAACGCTAGCGGADsg1 CAAR EC1-4 Amino Acid Sequence (Extracellular Portion up toGS-Linker, Transmembrane and Cytoplasmic Domains Same as for Dsg3 CAAR)

(SEQ ID NO: 40) MALPVTALLLPLALLLHAARPGSEFRIQVRDYNTKNGTIKWHSIRRQKREWIKFAAACREGEDNSKRNPIAKIHSDCAANQQVTYRISGVGIDQPPYGIFVINQKTGEINITSIVDREVTPFFIIYCRALNSMGQDLERPLELRVRVLDINDNPPVFSMATFAGQIEENSNANTLVMILNATDADEPNNLNSKIAFKIIRQEPSDSPMFIINRNTGEIRTMNNFLDREQYGQYALAVRGSDRDGGADGMSAECECNIKILDVNDNIPYMEQSSYTIEIQENTLNSNLLEIRVIDLDEEFSANWMAVIFFISGNEGNWFEIEMNERTNVGILKVVKPLDYEAMQSLQLSIGVRNKAEFHHSIMSQYKLKASAISVTVLNVIEGPVFRPGSKTYVVTGNMGSNDKVGDFVATDLDTGRPSTTVRYVMGNNPADLLAVDSRTGKLTLKNKVTKEQYNMLGGKYQGTILSIDDNLQRTCTGTININIQSFGNDDRTNTEPNASGDsg1 CAAR EC1-5 Nucleotide Sequence (Extracellular Portion up toGS-Linker, Transmembrane and Cytoplasmic Domains Same as for Dsg3 CAAR)

(SEQ ID NO: 41) GAAGAAGAAGGGTCAGCCACTATGGCACTTCCAGTGACCGCTCTGCTCCTGCCACTGGCCCTGCTGCTCCACGCTGCCCGCCCGGGCAGCGAGTTCAGGATCCAAGTCAGGGATTATAATACTAAAAACGGTACCATCAAGTGGCATTCCATACGCAGGCAGAAAAGGGAGTGGATTAAGTTTGCTGCCGCGTGCCGGGAGGGTGAAGACAATAGCAAACGGAATCCCATTGCAAAGATACATAGCGATTGCGCTGCCAATCAGCAGGTTACATATCGAATCTCCGGCGTGGGGATTGACCAGCCTCCTTATGGCATTTTCGTCATTAACCAAAAGACTGGCGAGATAAATATCACATCAATTGTGGACCGGGAAGTGACGCCGTTTTTTATCATCTACTGTAGAGCTCTGAACTCCATGGGCCAGGATCTGGAAAGGCCACTGGAGCTGAGGGTCAGGGTCCTTGACATCAATGACAATCCCCCCGTCTTTTCCATGGCCACGTTCGCCGGACAGATTGAGGAAAATAGCAATGCCAATACACTGGTGATGATCCTGAACGCTACCGACGCTGACGAGCCGAATAATCTGAACAGTAAAATTGCTTTTAAGATCATTCGGCAGGAGCCATCAGACAGCCCAATGTTTATCATTAACAGAAACACCGGAGAGATCCGCACAATGAACAATTTCCTGGATAGGGAACAGTATGGACAGTATGCACTCGCTGTTCGGGGCTCCGACCGGGACGGTGGAGCTGATGGCATGAGTGCCGAGTGCGAGTGCAATATCAAGATACTCGACGTAAATGATAATATTCCATACATGGAACAGAGCTCTTACACTATCGAGATCCAGGAGAATACTCTCAACTCTAATCTTCTTGAAATTAGAGTGATTGATCTCGACGAGGAATTTTCTGCCAATTGGATGGCTGTCATCTTCTTTATTAGTGGTAACGAGGGTAACTGGTTCGAGATAGAAATGAATGAAAGGACAAATGTGGGAATCTTGAAGGTGGTTAAACCACTGGACTACGAAGCAATGCAATCACTCCAGCTGTCAATAGGCGTCAGAAATAAGGCGGAGTTCCATCACTCCATTATGTCCCAGTATAAATTGAAAGCCAGTGCCATAAGCGTAACCGTGTTGAACGTGATAGAAGGGCCTGTTTTTCGCCCTGGGTCCAAAACCTACGTTGTGACAGGAAACATGGGATCCAACGACAAAGTCGGCGACTTCGTCGCAACAGACCTGGACACCGGTCGCCCTTCCACAACTGTGCGGTACGTGATGGGAAACAATCCAGCCGACTTGTTGGCAGTCGATAGCAGGACAGGGAAGCTGACCCTTAAAAACAAGGTTACAAAAGAACAATATAACATGCTGGGCGGCAAATATCAGGGAACCATTTTGTCAATCGACGACAACCTGCAGCGCACGTGCACGGGGACGATCAACATCAACATCCAGAGCTTTGGGAATGACGATAGAACCAACACAGAGCCCAACACAAAGATCACCACCAATACTGGCCGACAAGAATCCACCTCCAGCACAAACTATGATACGTCCACTACCAGTACAGACTCCAGTCAGGTTTACAGCAGTGAACCCGGTAATGGTGCCAAGGATCTCCTGAGTGATAATGTTCATTTTGGACCCGCTAGCGGADsg1 CAAR EC1-5 Amino Acid Sequence (Extracellular Portion up toGS-Linker, Transmembrane and Cytoplasmic Domains Same as for Dsg3 CAAR)

(SEQ ID NO: 42) MALPVTALLLPLALLLHAARPGSEFRIQVRDYNTKNGTIKWHSIRRQKREWIKFAAACREGEDNSKRNPIAKIHSDCAANQQVTYRISGVGIDQPPYGIFVINQKTGEINITSIVDREVTPFFIIYCRALNSMGQDLERPLELRVRVLDINDNPPVFSMATFAGQIEENSNANTLVMILNATDADEPNNLNSKIAFKIIRQEPSDSPMFIINRNTGEIRTMNNFLDREQYGQYALAVRGSDRDGGADGMSAECECNIKILDVNDNIPYMEQSSYTIEIQENTLNSNLLEIRVIDLDEEFSANWMAVIFFISGNEGNWFEIEMNERTNVGILKVVKPLDYEAMQSLQLSIGVRNKAEFHHSIMSQYKLKASAISVTVLNVIEGPVFRPGSKTYVVTGNMGSNDKVGDFVATDLDTGRPSTTVRYVMGNNPADLLAVDSRTGKLTLKNKVTKEQYNMLGGKYQGTILSIDDNLQRTCTGTININIQSFGNDDRTNTEPNTKITTNTGRQESTSSTNYDTSTTSTDSSQVYSSEPGNGAKDLLSDNVHFGPAS G

The presence of the construct encoding sequences in the plasmids wereconfirmed by digestion with SalI and BamHI. All constructs were verifiedby Sanger sequencing and the plasmids were purified in larger scale withremoval of endotoxins (qiagen endofree maxiprep).

Transient Expression

To test the expression of the CAAR constructs, 293T/17 cells weretransiently transfected using Polyethylenimine (PEI, jetPEI, polyplus)at a DNA:PEI ratio of 1:2. Expression was validated by flow cytometrywith anti-Dsg3 EC1-IgG1 (clone: Px43) and anti-human Fc-PE (cloneHP6017) after 36 hours on a LSRII flow cytometer (BD).

Production of HIV-1 Based Self-Inactivating Lentivirus

To facilitate stable expression of the CAAR constructs, VSV-Gpseudotyped lentiviral particles were produced using a 3rd generationpackaging system. Briefly, 293T/17 cells (ATCC CRL-11268) weretransfected at a confluency of 90% with a mixture of thepRRLSIN.cPPT.EF1a-Dsg3CAAR. WPRE plasmid, the envelope plasmid pMD2.G(addgene 12252), the packaging plasmids pRSVRev (addgene 12253) andpMDLgm/pRRE (addgene 12251) in a complex with Lipofectamine2000 (lifetechnologies). Lentivirus containing supernatant was harvested after 24,48 and 72 hours, filtered through a 0.4 micrometer membrane,concentrated at 12000 g for 12 hours at 4° C. and stored at −80° C.until further usage.

Reporter Assay with NFAT-GFP Jurkat T Cells

Jurkat cells were cultured at 37° C. with 5% CO2 in a completelyhumidified environment using RPMI1640, HEPES 10 mM,Penicillin/Streptomycin 1% and FBS at 10%. Hybridoma medium wasadditionally supplemented with 1% non-essential amino acids, 1% sodiumpyruvate and 0.5 mM BME. To test signal transduction by CAAR-targetinteraction, the CAAR constructs were expressed in a Jurkat reportercell line that has been selected (G418) for stable expression of GFPcontrolled under an NFAT response element, facilitating GFP expressionafter CAAR engagement and PLCgamma and IP3 mediated intracellularcalcium release. The Jurkat cell line was provided by Arthur Weiss(UCSF). Jurkat cells were transduced with CAAR lentivirus at amultiplicity of infection of 5-10 and expression of the CAAR constructwas validated after >72 hours with anti-ECS-Dsg3 mouse IgG1 (clone:5G11) and anti-mouse IgG1-APC (clone: A85-1, BD Pharmingen) by flowcytometry. To create target structures, tosylactivated dynabeads (lifetechnologies) were loaded with monoclonal human or mouse IgG1 specifcfor Dsg3 or with mesothelin (negative control) according to themanufacturer's recommendations. Additionally, serum from a PV patientand a non-PV individual were loaded onto beads. The AK23 hybridoma cellline (U.S. Pat. No. 7,550,562 B2) served as cellular target thatsecretes anti-Dsg3 antibodies and is surface-positive for theseantibodies. As negative control we used another hybridoma cell line thatsecretes antibodies against the human VH3-15 framework region (BK-2;U.S. Pat. No. 5,738,847 A) For characterization of CAAR-targetinteraction, the CAAR Jurkat cells were incubated for 4 hours witheither beads at a bead:cell ratio of 3:1 or target cells at increasingconcentration at 37° C. GFP expression was validated by flow cytometry.In addition to the beads and target cells, human B cells from a non-PVindividual and primary human keratinocytes (provided by the SDRC, coreB, University of Pennsylvania) were used to test for off-target effects.

Stimulation and Expansion of Primary Human T Cells

Primary human T cells were cultured in RPMI1640, 10% FBS and 10 mMHEPES, supplemented with 1% penicillin/streptomycin. T cells wereisolated from voluntary healthy donors and provided by the humanimmunology core (University of Pennsylvania). Bulk T cells (CD4+ andCD8+) were stimulated with anti-CD3 and anti-CD28 beads (dynabeads, lifetechnologies) at a bead:cell ratio of 3:1. When only CD8 cells wereused, the culture medium was supplemented with 150-300 IU/ml IL2. 24hours after stimulation, 10⁶ T cells were transduced with the CAARconstructs or a mock control at a MOI of 5-10. As mock control we usedan scFv-based chimeric antigen receptor against human CD19 or humanmesothelin. Expansion of the T cells was monitored for 8-14 days withanalysis of cell density and cell volume every 2nd day for the first 6days, after that daily. Cell volume and cell density was analyzed with aCoulterCounter (Beckman Coulter). Killing assays were performed at acell volume of ˜400 fl. Cell surface expression of the CAAR constructswas validated by flow cytometry using anti-Dsg3 antibodies (clones: Px43(IgG1), Px44 (IgG1), F779 (scFv), AK23 (mouse IgG1)) and detectionantibodies against human IgG-FC (clone: HP6017), mouse IgG1 (clone:A85-1) or HA peptide (clone: 3F10). ScFv-based chimeric antigenreceptors were detected with polyclonal donkey anti-human IgG (heavy andlight chain) or goat anti-mouse IgG.

In Vitro Killing Assay

In vitro killing was tested with a 51Cr-release assay. 5×105 targetcells were loaded with 50 microCi of Na2 51CrO4 (Perkin Elmer) for 90minutes, washed twice and resuspended in phenolred-free medium with 5%FBS. CAAR or mock transduced T cells were coincubated with loaded targetcells for 4 and 24 hours at various effector: target ratios and chromiumrelease into the supernatant was measured with a microbeta 2 platecounter (Perkin Elmer). When only CD8 cells were used, the assay wasperformed in presence of 75 IU/ml IL2. Spontaneous release by targetcells only was analyzed in the same volume and maximum release wasassessed by treating target cells with SDS at a final concentration of2.5%. To test redirected, Fc-receptor mediated lysis, K562 cellspositive for CD32 were incubated with CAAR T cells in the presence ofhuman monoclonal anti-Dsg3 IgG1 (clone: PV2B7) at a concentration of 5micrograms/ml or anti-human CD3 (okt3) at the same concentration.

Specific lysis was analyzed as follows:Percent Specific Lysis: [(Experimental Release−SpontaneousRelease)/(Maximum Release−Spontaneous Release)]*100

In Vivo Efficacy Testing of CAAR T Cells

CAAR or control-CAR transduced T cells were expanded as describedherein. For in vivo experiments, CAAR or control-CAR T cells wereadjusted to a concentration of 3×10⁷ cells/ml and mixed withGFP-clickbeetle red or green transduced AK18,19 or 23 hyrbidoma cells(at 10⁶ cells/ml), resulting in a T cell to target ratio of 30. Cellswere kept on ice and 200 μl of the cell mixture was injected into thetail vein of NSG mice, resulting in 3×10⁶ T cells and 10⁵ target cellsper mouse. After i.v. injection, NSG mice were injected with D-Luciferinmonopotassium salt solution at a dose of 300 mg/kg body weight.

Bioluminescence was quantified with a PerkinElmer IVIS spectrumpreclinical in vivo imaging system. Additonal assessment of tumor burdenby bioluminescence was done on day 3, 7, 13, 17/18, 26 and 35 afterinjection. Analysis was done with LivingImage software 4.4. Foranalysis, rectangle-shaped regions of interests with identical areaswere set up from the head of the mouse to the middle the tail. Totalflux in photons/second was calculated after background luminescencesubtraction. A bioluminescence of 10⁸ photons/second was used to declarethe mice dead, since this represented a ˜100 fold expansion of theinitial tumor burden and indicated loss of tumor control.

Mice were sacrificed in accordance to an approved IACUC protocol. Spleenand bone marrow samples were kept in RPMI medium supplemented with 10%FBS until further processing. Blood samples from sacrificed mice wereobtained by cardiac puncture and anticoagulated with EDTA. Single cellsuspension from spleen and flushed bone marrow samples were obtained bypassing cells through a 100 um cell strainer. 10⁶ Cells were stainedwith anti-human CD3 (clone Okt3), anti-human CD45 (clones HI30 or 2D1)and anti-human Dsg3 (clone Px44, no loss of binding by EDTAdenaturation) for 25 minutes at room temperature, fixed with BD FacsLyse stored at 4 degrees Celsius and analyzed on a BD LSRII flowcytometer.

The results of the experiments are now described.

Pemphigus vulgaris (PV) is an antibody mediated autoimmune diseasecausing potentially fatal blistering of the skin and mucous membranes.It is a potentially life threatening due to malnutrition, infection, anddehydration. PV is a model tissue-specific, antibody-mediated autoimmunedisease because the autoantigen Dsg3 (desmoglein 3) is well-defined andanti-desmoglein antibodies are necessary and sufficient to causecharacteristic suprabasal blisters in animal and human skin models.

Autoantibodies are synthesized and secreted by autoreactive Blymphocytes and primarily target the extracellular EC1-3 domains of Dsg3where trans- and cis-adhesive residues are located. The most effectivetreatment strategies in PV target B lymphocytes and include systemiccorticosteroids, azathrioprine, mycophenolate mofetil, andcyclophosphamide to inhibit lymphocyte proliferation. Rituximab is usedas an anti-CD20 B cell depletion mechanism via a B lymphocyte specificsurface molecule. However, there is no treatment that targets onlyautoreactive cells as opposed to all B cells. This results in currenttreatment strategies having severe side effects, including fatalinfection and secondary cancers.

Recently, genetically engineered T cells expressing a chimeric antigenreceptor (CAR) against the B cell surface marker CD19 (D L Porter et al,NEJM 2011; S A Grupp et al, NEJM 2013) has been found to specificallytarget and kill CD19+ B cells and can induce long-lasting remission inpatients with refractory B-cell malignancies.

As described herein, T cells can be engineered to kill target cellsindependent of MHC and co-stimulatory signals by expressing arecombinant chimeric T cell antigen receptor with an extracellulardomain that specifically recognizes the target antigen, and anintracellular domain that is sufficient to activate signaling afterantigen binding (Chimeric Antigen Receptors, or CARs). Chimeric AntigenReceptors consist of customized cell surface receptor (typically anantibody against a specific cell surface molecule on the target cell),transmembrane domain and intracellular domains of costimulatorysignaling receptors in the same protein.

The advantages of using CARs include the ability to be directed againstvirtually all known antigens, CARs act independently of MHC expressionof the target cell, and CAR binding to its target antigen results inactivation of the T cell independently of costimulatory signals from thetarget cell. Engineering T cells for PV treatment relies on the perfecttarget for a genetically engineered T cell that is shared among alltarget cells, as well as unique to the target cell. For example,autoreactive B cells in PV express a surface Ig that binds to desmoglein3.

The design for the genetically engineered T cells for PV optimallyincludes a typical chimeric antigen receptor (CAR) with a high affinityantibody binding moiety that targets specific autoantibodies. Inautoimmune diseases such as PV, the pathogenic cells (B cells) alreadyexpress the high affinity autoantibody on their cell surface. Thus,genetically engineered T cells for autoantibody-mediated diseases shouldexpress the antigen, not the antibody, on its cell surface=a chimericautoantibody receptor (CAAR). In the case of PV, this is Dsg3.

FIG. 1 is a schematic drawing that depicts how the proposed chimericautoantibody receptor (CAAR) is distinct from all previously developedtechnologies. The left half of the figure shows a chimeric antigenreceptor (CAR) on an effector cell to(the patient's own T cells), whichtargets an antigen (CD19) that is specifically expressed on the B celllymphoma. What makes PV autoreactive B cells unique from all other Bcells is that they express an autoantibody on their cell surface that isspecific for the disease autoantigen, desmoglein 3 (Dsg3). Hence, the PVCAAR is the autoantigen (Dsg3), which targets the Dsg3-autoantibodyexpressed on the surface of autoreactive PV B cells. FIG. 2 illustratesthat the interaction between engineered chimeric T cell receptors andtarget Dsg3 specific B cells is more specific for PV than CD19- orCD20-targeted therapies. Unlike a CD19- or CD20-targeted therapy, ageneralized immune suppression should not occur with a Dsg3 targetedtherapy. Engineered T cells are more sustainable than monoclonalantibody-based therapy because T cells proliferate in response toantigen and form memory T cells. Moreover, engineered chimeric T cellreceptors that target Dsg specific B cells remove both memory andshort-lived antibody-secreting B cells (FIG. 3 ).

Desmogleins are ideal autoantigens for a chimeric autoantibody receptor(CAAR) because they consist of modular extracellular domains that can betruncated. Also, CAAR efficiency is influence by the intermembranedistance between the T cell and its target, see FIG. 4 .

FIGS. 5-6 illustrate the ability to amplify individual domains of Dsg3(FIG. 5 ) and CD137 (FIG. 6 ) from cDNA of peripheral blood mononuclearcells. FIG. 7 further shows amplification of Dsg3 and Dsg3 CAAR fromplasmid DN653.

Dsg3 CAAR protein was analyzed by western blot 48 hours aftertransformation and cell lysis of 293T cells under reducing conditions(FIG. 8 ). Dsg3 E-His baculovirus supernatant was a positive control,untransfected HEK293T cells were a negative control. The expected sizewas 96 kDa for the unglycosylated protein, which typically migrates at˜112 kD with glycosylation.

To evaluate the cytotoxicitiy of Dsg3 CAARs, the ability of Dsg3 CAARexpressed in primary human T cells to kill target cells expressinganti-Dsg3 surface autoantibodies (test for efficacy) and the potentialfor Dsg3 CAAR to kill off-target cells that may express a surface Fcreceptor, which could bind PV autoantibodies and result in unintendedredirected lysis (test for safety) was determined.

Dsg3 CAAR specificity toward intended and unintended targets was testedin lysis assays. It was anticipated that the Dsg3 CAART cells would killanti-Dsg3 B cells as an intended target because of a high affinityinteraction between the Dsg3 CAAR and anti-Dsg3 autoantibody (left sideof FIG. 9 ). Whereas weak hemophilic interactions between Dsg3 CAARTcells and cells expressing anti-Dsg3 with a Fc receptor (keratinocytes)would not result in killing by the Dsg3 CAART cells (right side of FIG.9 ).

As expected, Dsg3 CAAR Jurkat cells did not show strong redirectedlysis. NFAT-GFP Jurkats cells expressing Dsg3-CAAR were stimulated withantibody coated beads at a ratio of 3:1 (beads:cells). Flow cytometryplots shown in FIG. 10 indicated that signaling in Dsg3 CAAR Jurkatcells was present after exposure to PV target antibodies. AK23, PV4B3,and PV2B7 are Dsg3-specific mAbs, which if bound to the CAAR Jurkatcells, should trigger signaling that induces GFP expression. EF1apromoter functioned better than the PGK promoter and resulted inspecific signaling. SS1=anti-mesothelin CAR was used as a positivecontrol and had baseline positive activity. Non-transduced cells wereused as a negative-control and no GFP signal was detected.

Low level, but specific, signaling was induced in Dsg3 CAAR Jurkat cellsafter exposure to polyclonal pemphigus vulgaris (PV) patient serum IgG(reflecting the low overall percentage of total IgG that isDsg3-specific) (FIG. 11 ). Dsg3 CAAR Jurkat cells also responded to lownumbers of surface IgG on cells (AK23 hybridoma) in a dose-dependentmanner. Signaling was not induced when Dsg3 CAAR Jurkat cells wereexposed to Dsg3 expressing keratinocytes, indicating that interactionsof Dsg3 with desmosomal cadherins on keratinocytes should not result inskin or mucous membrane toxicity.

To test safety of Dsg3 CAAR effector cells, different scenarios wereproposed for testing cytotoxicity toward target cells that expressanti-Dsg3 surface autoantibodies and off-target cells that expresssurface Fc receptors that could bind serum PV autoantibodies resultingin unintended redirected lysis. The cells on the left in FIG. 14 showexpected killing of anti-Dsg3 B cells as the intended target. The cellson the right in FIG. 14 show unintended killing of cells that express Fcreceptors, potentially through redirected lysis.

Dsg3 CAAR effector cells were exposed to a K562 cell line that expressedsurface Fc receptors pre-loaded with PV anti-Dsg3 mAb (PV2B7). Noredirected lysis was observed with PV mAb bound to Fc receptor (leftgraph in FIG. 15 ), as it behaved similar to the negative controls andnon-transduced cells (right graph in FIG. 15 ).

TCR activation (and hence killing) is dependent on the distance betweeneffector and target cell (ideal distance is 14-15 nm). Shorter or longerdistances will result in loss of TCR activation. The target (surfaceIgG) for the Dsg3 CAAR is ˜8.4 nm. Desmoglein 3 is roughly 12.5-18 nmlong. The desmosomal gap is ˜40 nm. Desmoglein 3 consists of 5 Ig-likedomains with a size of approximately 3.5 nm each. Trans- andcis-interactions are approximately 24.5 nm when interacting through EC2cis-interaction.

To determine if expression of just a part of Dsg3 may result in enhancedCAAR activation, due to optimal intercellular distance for theimmunologic synapse, different Dsg3 EC domain constructs (intermolecularadhesion domains are contained in EC1-2) were used. Other EC-domainconstructs may also be used and are detailed in the disclosure. FIG. 16Ais an image of an electrophoretic gel showing amplification of thedifferent Dsg3 extracellular domains, EC2-3, EC1-2, EC1-3, EC1-4 andEC1-5 (FIG. 16B), which were constructed to optimize Dsg3 CAARcytotoxicity, since the efficacy of CAAR-mediated cytotoxicity isdependent on the distance between effector and target cell.

Dsg EC1-3, EC1-4, EC1-5 CAARs were expressed in primary human T cellsand recognized by 3 different PV anti-Dsg3 mAbs, AK23, Px44, and F779(FIG. 17 ). EC1-2 did not effectively express.

The efficacy of the Dsg3 CAAR against an anti-Dsg3 IgG mouse hybridoma(meant to model a PV-specific human memory B cell or plasmablast thatdisplays anti-Dsg3 IgG on the cell surface) is shown in FIG. 18 . TheDsg3 CAAR was expressed on the surface of primary human T cells andspecific in vitro killing of AK23 (an anti-Dsg3 hybridoma) was observedin a chromium release assay after 4 hours as compared to the negativecontrol, BK2.

Efficacy of Dsg3 CAAR T cells was further shown to specifically killAK23 hyridoma in a chromium-51 release assay. Dsg3 CAAR killing of AK23hybridoma increased over time in a chromium release assay after 24 hours(FIG. 19 ). Essentially, about 100% killing was observed with the Dsg3EC1-3 and EC1-4 CAAR after 16 hours. The killing efficacy of the CAARscorrelated with CAAR size (shorter was better EC1-3>EC1-4>EC1-5). HumanCD19 was not expressed on the target hybridoma cell as anti-human CD19CAR was the mock control. Control hybridoma (BK2), which does notexpress a Dsg3-autoantibody, demonstrated some killing by Dsg3 CAARcells over 24 hours, perhaps due to human-mouse alloreactivity.

Dsg3 CAART cells killed anti-Dsg3 cells targeting a broad range ofepitopes, F779 (anti-EC1) and PVB28 (anti-EC2). Dsg3 EC1-4 CAAR(EC1-4bbz) killed anti-EC1, anti-EC2 and anti-EC3 B cells, see FIG. 20 .Dsg3 EC1-4 CAAR also did not kill K562 wild type (wt) cells (FIG. 21 ),as well as non-transduced T cells did not kill F779/PVB28 K562 cells.The K562 cells expressing F779 or PVB28 surface immunoglobulins alsoexpressed CD19 and mesothelin. Nontransduced (NTD) T cells were used asa negative control.

To determine if antibody density is comparable between hybridomas andmemory B cells, quantification of anti-Dsg surface IgG was performed onhybridoma cells, AK18, AK19 and AK23, and human B cells. Density of IgGon hybridomas was measured by fluorescence/protein (F/P) ratio. Giventhe large size differences between the cells, hybridoma cell about 567.5um² and human B cells about 160.3 um², there was about 3.54× moresurface area for hybridoma than human memory B cells. Thus, thefluorescence/protein (F/P) ratio needed to take the surface area intoconsideration. The normalized surface IgG density was calculated atabout AK18: 1130, AK19: 5300, AK23: 1765 and human B cells: 3570, seeFIG. 22 .

Dsg3 CAART cells were further tested to determine if antibody affinitiesaffect efficacy. The relative affinities of the target immunoglobulinssecreted by the hybridomas used in the killing assays described herein,AK18, AK19 and AK23, are shown in FIG. 23 . The three antibodies havevarying affinities and Dsg3 CAART cells are effective against all three.

To further test the efficacy of Dsg3 CAART cells, soluble blockinganti-Dsg3 antibody was added to the killing assay. Dsg3 CAART cellsdemonstrated specific killing even in the presence of increasingconcentrations of soluble antibody (FIG. 24 ). The killing assays wereperformed in the presence of the hybridoma and soluble antibody from thesame clone. FIG. 24 demonstrates that Dsg3 CAART cells targeted cellseven in the presence of soluble blocking anti-Dsg3 antibody for allconditions tested.

To test off-target killing of Dsg3 CAART cells, potential scenarios wereexamined. Cells expressing Fc receptors that bind serum anti-Dsg IgGcould be killed by T cells expressing the Dsg CAAR. However, theintermembrane distance is likely too large to allow for effectivekilling. Serum anti-Dsg3 IgG typically also only presents a minority(˜1%) of total serum IgG, suggesting that toxicity should be minimal.Keratinocytes that express desmosomal cadherins (desmogleins anddesmocollins) could theoretically interact with the Dsg CAAR and bekilled. However, intermembrane distance is likely suboptimal forkilling, and affinity of interaction is too low (μM range).

Dsg3 bearing T cells were tested in a reversed antibody-dependentcellular toxicity (rADCC) assay to determine if Dsg3 CAART cellstargeted Fc receptor expressing cells (upper graph in FIG. 25 ). FcgRIexpressing K562 cells (CD64+ K562) were co-incubated for 8 hours withCAART cells in PV serum. Target cells were 100% positive for surfaceIgG, yet were not killed by Dsg3 CAART cells. K562 cells expressingPVB28 IgG4 in normal medium were used as a positive control to show thatthe same CAART cells were, in fact, functional (lower graph in FIG. 25), i.e. specifically killed target cells.

Dsg3 CAAR T cells were incubated with primary human epidermalkeratinocytes grown in calcium-containing media to induce desmosomeassembly. No killing was observed with the Dsg3 EC1-4 CAART cells (FIG.26 ). In contrast, CART cells consisting of a mAb against Dsg3 and Dsg1effectively killed keratinocytes.

Dsg3 CAAR T cells also effectively controlled IgG secreting hybridomacells in vivo. FIG. 27 shows AK19 hybridoma cells and either Dsg3 EC1-4CAAR T cells or control CAR T cells were co-engrafted into NSG mice. Atthe indicated time points, tumor burden was quantified bybioluminescence imaging. Bioluminescence>10e8 (total flux[P/s]) was usedto declare mice ‘dead.’

In escape mice (the 4 CAAR treated mice that had delayed outgrowth ofAK19), the recovered AK19 cells were mostly surface immunoglobulinnegative, indicating effective elimination of target (anti-Dsg3 IgG+)cells (FIG. 28 ). AK19 hybridomas were labeled with GFP and click beetlegreen luciferase prior to in vivo injection, which allowed thedetermination of whether the AK19 cells remained sIg+. 1e6 bone marrowcells were stained with saturating amounts of anti-mouse IgG1-APC.41.6+49.6=91.2% of cultured AK19 hybridoma cells were sIg+ (left panel).6/8 co-injected mice showed a pattern like 9382, with few detectableGFP+ cells. 9406 and 9407 (escape mice) showed GFP+ cells that hadreduced or no surface IgG expression.

Dsg3 CAAR T cells further engrafted and maintained long-term CAARexpression (FIG. 29 ). In fact, significant expansion of the cells wasobserved as compared to control CAR T cells. Percent of human T cells inmouse bone marrow (BM):control CAR versus Dsg3 CAAR

FIGS. 30A-C show the presence of Dsg3 CAAR T cells that engrafted indifferent immunological compartments, blood (FIG. 30A), bone marrow(FIG. 30B), and spleen (FIG. 30C).

Shown in FIG. 31 is the complete blood count from 7 CAAR treated mice 35days after CAAR injection. No depletion of Fc-receptor bearing cells wasdetected (NE=neutrophils, MO=monocytes). In vivo efficacy of the Dsg3EC1-4 CAAR and Dsg3 EC1-3 CAAR was shown against AK23 (anti-EC1).

Dsg3 EC1-3 CAAR further reduced AK23 tumor burden by bioluminescence(FIG. 32A) and increased survival (FIG. 32B). Potentially similar toAK19, mice that escape the Dsg3 EC1-3 CAAR may be selected for negativesurface immunoglobulin AK23 cells. FIGS. 33-35 further show that Dsg3EC1-4 CAAR T cells show efficacy against a broad range of targets invivo, including AK18 (anti-EC3), AK19 (anti-EC2), and AK23 (anti-EC1),as evidenced by decreased bioluminescence in Dsg3 CAART-versus controlCART-treated mice and increased survival of Dsg3 CAART-treated mice.

In summary, a Dsg3 CAAR has been developed that shows specific binding,activation by, and killing of cells expressing surface anti-Dsg3 IgG(efficacy). The Dsg3 CAAR does not activate in response to keratinocytesexpressing Dsg3, or cells expressing Fc receptors that may bind serumanti-Dsg3 IgG (safety). Furthermore, the Dsg3 CAAR is a novel andspecific strategy to target only the autoreactive B cells in PV andcould be used as a proof of principle for the therapeutic use of CAARsin other autoantibody-mediated diseases.

To test if CAAR cells can be engineered with Dsg1 and be as effective atkilling as Dsg3 CAAR cells, a CAAR construct including Dsg1 wasgenerated (FIG. 36 ). Based on results optimizating the Dsg3 CAAR, aDsg1 CAAR was constructed consisting of EC1-5 domains of Dsg1 for theCAAR extracellular domain. K562 cells were engineered to expressmonoclonal surface IgG with Dsg1 EC1 or Dsg1 EC2 specificities. After 16hrs in a ⁵¹Chromium release assay, Dsg1 CAAR cells effectively killedanti-EC1 and anti-EC2 B cells, see FIG. 37A, but did not kill wild typeK562 cells or K562 cells expressing anti-Dsg3 antibodies (FIG. 37B).

To test if Dsg1 or Dsg3 CAARs engineered with KIR domains are effectiveat killing target cells, a CAAR construct including KIR transmembraneand cytoplasmic domains was generated (FIG. 38 ). Based on resultsoptimizating the Dsg3 CAAR, a Dsg3 KIR CAAR was constructed consistingof EC1-3 or EC1-4 domains of Dsg3 with KIR transmembrane and cytoplasmicdomains. PVB28 cells express anti-Dsg3 antibodies. After 16 hrs in a⁵¹Chromium release assay, Dsg3 KIRCAAR cells effectively killed anti-EC2B cells (FIG. 39A) and not control cells (not expressing anti-Dsg3antibodies), (FIG. 39B). The disclosures of each and every patent,patent application, and publication cited herein are hereby incorporatedherein by reference in their entirety. While this invention has beendisclosed with reference to specific embodiments, it is apparent thatother embodiments and variations of this invention may be devised byothers skilled in the art without departing from the true spirit andscope of the invention. The appended claims are intended to be construedto include all such embodiments and equivalent variations.

What is claimed:
 1. A method of treating pemphigus vulgaris, paraneoplastic pemphigus, or pemphigus foliaceus in a subject in need thereof, the method comprising administering to the subject a genetically modified cell comprising a chimeric receptor comprising an extracellular domain comprising Dsg1, Dsg3, or a fragment thereof that binds an autoantibody expressed on a B-cell in the subject, and a transmembrane domain, wherein the cell expresses the chimeric receptor and binds the autoantibody expressed on the B-cell or induces killing of the B-cell expressing the autoantibody.
 2. The method of claim 1, wherein the genetically modified cell is selected from the group consisting of a helper T cell, a cytotoxic T cell, a memory T cell, a regulatory T cell, a gamma delta cell, a natural killer cell, a cytokine induced killer cell, and a cell line thereof.
 3. The method of claim 1, wherein the extracellular domain comprises Dsg3 or a fragment thereof.
 4. The method of claim 3, wherein the extracellular domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, or an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:36.
 5. The method of claim 1, wherein the chimeric receptor further comprises a CD8 alpha chain signal peptide.
 6. The method of claim 5, wherein the CD8 alpha chain signal peptide comprises the amino acid sequence of SEQ ID NO:1.
 7. The method of claim 1, wherein the extracellular domain comprises Dsg3 or a fragment thereof and the chimeric receptor further comprises a propeptide of Dsg3.
 8. The method of claim 1, wherein the transmembrane domain comprises a KIR transmembrane domain.
 9. The method of claim 1, wherein the transmembrane domain comprises a CD8 alpha chain hinge and transmembrane domain.
 10. The method of claim 9, wherein the CD8 alpha chain hinge and transmembrane domain comprises the amino acid sequence of SEQ ID NO:13.
 11. The method of claim 1, wherein the chimeric receptor further comprises a peptide linker.
 12. The method of claim 11, wherein the peptide linker comprises the amino acid sequence of SEQ ID NO:14.
 13. The method of claim 1, wherein the chimeric receptor further comprises a KIR cytoplasmic domain.
 14. The method of claim 1, wherein the chimeric receptor further comprises an intracellular signaling domain.
 15. The method of claim 14, wherein the intracellular signaling domain comprises a CD137 intracellular domain.
 16. The method of claim 15, wherein the CD137 intracellular domain comprises the amino acid sequence of SEQ ID NO:15.
 17. The method of claim 14, wherein the intracellular signaling domain comprises a CD3 zeta signaling domain.
 18. The method of claim 17, wherein the CD3 zeta signaling domain comprises the amino acid sequence of SEQ ID NO:16.
 19. The method of claim 1, wherein the genetically modified cell has limited toxicity toward a healthy cell.
 20. The method of claim 1, wherein the subject has pemphigus vulgaris.
 21. The method of claim 1, wherein the subject is a human. 